Thermo Fisher Scientific REMBRANDT DISH and AP Mode d'emploi

Marque: Thermo Fisher Scientific

Modèle: REMBRANDT DISH and AP

Taper: Mode d'emploi

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In situ Hybridisation and Detection

DISH & AP Detection Kit-V11

DISH-AP kit for

the detection of

Biotin Label

product code

Digoxigenin label

product code # Assays

HPV screening

A100K.0105

A100K.9905

10-100

HPV typing

A103K.0105

A103K.9905

10-100

PanPath

Instraat 5b3

6021 AC Budel

The Netherla nds

Tel. +31 (0) 495 499090

E-mail info@panpath.nl

Web site www.pa npat h. nl

For research use only RUO

REMBRANDT®

KIT MANUAL REMBRANDT® DIS H & AP DETECTION

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Contents

page

Chapter 1 Introduction 03

1.1 Intended use 03

1.2 The ISH principle 03

1.3 Controls 03

1.4 Conte nts of the REMBRANDT® kit 03

1.5 Materials required but not included 04

1.6 Storage and shelf life 04

1.7 Safety precautions 04

1.8 Performance precautions 05

1.9 Preparations of reagents in advance 05

1.10 Preparation of the proteolytic work solution 05

Chapter 2 REMBRANDT® DISH & AP Detection protocol 06

2.1 Specimen collection and pre-treatment 06

2.2 Proteolytic treatment 06

2.3 Hybridisation procedure 07

2.4 Detection and staining procedure 07

2.5 Counter stain procedure 07

Chapter 3 Limitations of procedure 08

3.1 Limitations 08

3.2 Interpretation of results 08

Chapter 4 References 10

Chapter 5 Probe specifications 11

Chapter 6 Trouble shooting guide 13

6.1 Introduction 13

6.2 No section or cells left on the slides 13

6.3 Weak or no staining on a suspected positive sample 14

6.4 Negative staining of the positive control 14

6.5 Positive staining of the negative control 15

6.6 Non-specific background staining 15

6.7 Cross hybridisation 15

Chapter 7 Language Reference Guides 16

7.1 English & German 16

7.2 Fre nc h & Spanish 17

7.3 Dutch & Italian 18

7.4 Greek 19

Immaterial property information 20

CHAPTER 1 – INTRODUCTION

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Chapter 1 Introduction

1.1 Intended use

REMBRANDT® has been designed for the detection of specific DNA or RNA sequences by using

the in situ Hybridisation (ISH) technique in paraffin embedded tissue sections, cytological

specimens and frozen sections.

1.2 The ISH principle

ISH enables the detection of specific DNA or RNA sequences in histological and cytological

specimens, without losing the often very essential morphological details. The principle of ISH is

based on a “reaction” (= hybridisation) between a specifically labelled DNA or RNA sequence (=

probe) and a DNA or RNA sequence present in the sample (= target). In case of matching

sequences, a hybrid between the probe and target will be formed. Non-perfect matches are washed

out by the stringency wash procedure (PanWash). The formed hybrids can easily be visualised by a

specific staining procedure, i.e. substrate conversion by enzyme-conjugated antibodies. This

conversion, i.e. the combination of NBT/BCIP and Alkaline Phosphatase (AP) conjugated anti-

DIG or anti-BIO antibodies provided with this kit, will yield a detectable and coloured

precipitation. The ISH technique is highly sensitive, specific, fast and easy to perform. Moreover,

no radioactivity is involved. The reagents supplied with this kit are tailored to each other and

therefore, REMBRANDT® is the ultimate user-friendly tool for performing ISH.

1.3 Controls

Use of both positive and negative controls is an essential part of the routine. To ensure that the ISH

procedure is performed correctly and that observed positive and/or negative staining are specific,

controls should be included in each experiment. This REMBRANDT® kit includes positive and

negative control probes serving as a procedure control to be used on sections from the specimen

under investigation. The positive control slides contain the desired target DNA and serve as a

control for the specific probe. Additional control slides and probes are available from PanPath;

please contact your local supplier.

1.4 Contents of a REMBRANDT® DISH & AP Dectection Kit

Item label description

Item (cap) colour

Item contents description

Item amount

Transparant vial

: Pepsin digestion reagent

1 gram

Transparent vial

: Pepsin diluent (1M HCl solution)

15 mL

Yellow/Purple vial

: Specific1 BIO or DIG labelled DNA probe(s)

1 mL

│

Pink vial

: DISH positive control oligo probe (BIO or DIG)

1 mL

│

Green vial

: DISH negative control DNA probe(BIO or DIG)

1 mL

White vial

: PanWash (differentiation reagent)

2 x 15 mL

Red vial

: AP-conjugated anti-DIG or anti-BIO

15 mL

Blue vial

: NBT/BCIP substrate

15 mL

Orange vial

: Nuclear Fast Red counterstain

15 mL

White pouches

: TBS buffer salt

2 pcs

PEPSIN POW

PEPSIN DIL

…...2

…...2 DISH

GC% ….

2 50%

……

2 AP

NBT/BCIP

NFR

TBS

DIGEST

PROBE

ONTROL

CONTROL

PANWASH

CONJ

SUBS

COUNT

WASH

CHAPTER 1 – INTRODUCTION

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1.5 Materials required but not included

− Xylene for dewaxing paraffin sections.

− Fixative for cytological and frozen specimens.

− Distilled or deionised water.

− 100% Ethanol.

− 95% Ethanol.

− 70% Ethanol.

− Water-based mounting medium.

− Pipettes and tips to deliver 10-1000 µL.

− Incubation oven, set at 56-60°C to bake paraffin sections.

− Heating block/slide warmer, set at 37°C.

− Surface thermometer.

− Hotplate, set at 95°C.

− Light microscope for objective 10-100x.

1.6 Storage and shelf life

− Store all reagents at 2-8°C upon receipt of the kit.

− Store the dissolved and aliquoted pepsin reagent at -20°C, stable for at least 1 year when kept

frozen.

− Store the dissolved TBS buffer at 2-8°C when not in use.

− When used and stored as indicated, the kit is stable until the expiration date printed on the box.

1.7 Safety precautions

− Some reagents contain preservatives which can cause irritation when exposed to skin or mucous

membranes. The concentrations of these preservatives, however, are very low (< 0.1%). If

reagents come into contact with skin or eyes, rinse with large volumes of clean water.

− Never pipette solutions by mouth.

− The control slide in the kit contains pathogenic material fixed in 4% para-formaldehyde making

specimens non-infectious; however, we advise taking standard precaution measures for handling

infectious organisms.

1.8 Performance precautions

~ Kit contents continued ~

Item label description

Item (cap) colour

Item contents description

Item amount

White box A

: Coated + glass slides

50 pcs

White box B

: Coverslips

100 pcs

White box C

: Positive control slides

2 pcs

1 For specific probe specifications see chapter 5

2 Depends on Kit specification

GL SLIDES

COVERSL

……

SUPPORT

CONTROL

CHAPTER 1 – INTRODUCTION

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− Read all instructions before processing any assay.

− DO NOT use reagents beyond their expiry date.

− Allow all components to warm up to room temperature (20-25°C) before use.

− Homogenize probe solution before use.

− Avoid cross contamination of specimens.

− DO NOT substitute a reagent with one from another manufacturer.

− When using treated glass slides other than those provided in the kit, specimens may fall off during

the procedure.

− DO NOT perform the differentiation step on specimens incubated with the positive control oligo

probe (pink)!

1.9 Preparation of reagents in advance

Pepsin digestion reagent:

Dissolve the proteolytic reagent in 8 mL of distilled or deionised water (upon receipt of the kit).

Aliquot in portions of i.e. 150 µL and store at-20°C.

Pepsin diluent:

Dilute the 1M HCl solution (transparent) to the application required concentration (paraffin sections

0,1M; cytological and frozen preparations 0,01M) with distilled or deionised water.

TBS buffer salt:

Dissolve 1 pouch in 1000 mL distilled or deionised water. Dissolve the salt completely and keep the

buffer free from contamination.

1.10 Preparation of the proteolytic work solution

Prepare proteolytic work solution; 300 to 400 µL per section of 1 cm2. Make fresh work solution just

before use and discard non-used solution.

Paraffin sections:

dilute aliquoted proteolytic reagent 100x in 0.1M HCl, e.g. add 100 µL to 5 mL 0.1M HCl and mix.

Cytological specimens:

dilute aliquoted proteolytic reagent 25,000x in 0.01M HCl, e.g. add 8 µL to 100 mL 0.01M HCl and

mix.

Frozen sections:

Dilute aliquoted proteolytic reagent 50,000x in 0.01M HCl, e.g. add 4 µL to 100 mL 0.01M HCl

and mix.

CHAPTER 2 – REMBRANDT® DETECTION PROTOCOL

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Chapter 2 REMBRANDT® DISH & AP Detection Protocol

All incubation steps should be performed in a closed incubation chamber which contains a

liquid (water) creating a saturated moisturised environment. During the incubation steps,

evaporation of reagents should be prevented. Once the hybridisation procedure has been

started the specimen should not be allowed to dry.

2.1 Specimen collection and pre-treatment

Paraffin embedded tissue sections

A standard procedure for tissue fixation and embedding usually involves the use of formalin and

paraffin. The optimal tissue block size is 0.5 cm3. The formalin should be buffered and fixation times

should (preferably) not exceed 12 hours. Excess and/or insufficient fixation may yield suboptimal

morphology and target preservation. Embedding in paraffin should not exceed temperatures of 65°C.

Sample preparation: stretch 4 µm paraffin sections on distilled water of 55°C without any additives

and collect sections on bio-adhesive (i.e. organosilane) coated glass slides. Bake the slides at 56°C -

60°C in a dry air oven for 2-16 hours. Slides can be used immediately or they can be stored at room

temperature for up to 3 months. Prior to ISH, slides need to be dewaxed in fresh xylene for 2 x 10

minutes. Incomplete removal of formalin and/or paraffin may affect the result of the procedure.

Remove the xylene by placing the slides in 100% ethanol for 5 minutes. Air dry the slides for

approximately 5-10 minutes and start with proteolytic treatment.

Cytological specimens

Make sure that no multilayer of cells is formed when making a cytological specimen. A multilayer

will hamper microscopic examination of the result. The specimen be processed as soon as possible

after sampling.

Sample preparation: deposit cells on coated glass slides and air dry for 30 minutes. Fix the cells with

a cross-linking fixative (e.g. 4% para-formaldehyde) for 10 minutes at room temperature and rinse

with PBS. Dehydrate in graded ethanol series, air dry and start with proteolytic treatment.

Frozen sections

In general, small pieces of tissue (max. 1 cm3) are snap frozen in liquid nitrogen and either stored at -

70°C or used immediately. Frozen sections are more fragile than paraffin embedded tissue sections.

They should be handled with care and processed as soon as possible.

Sample preparation: collect frozen sections (4 µm) on bio-adhesive (i.e. organosilane) coated glass

slides and air dry for 30 minutes. Fix the sections with a cross-linking fixative (e.g. 4% para-

formaldehyde) for 10 minutes at room temperature. Dehydrate in graded ethanol series, air dry and

start with proteolytic treatment.

2.2 Proteolytic treatment

Place both test and control slides on a 37°C heating block or slide warmer and add 300-400 µL of a

freshly prepared, pre-warmed proteolytic work solution to each specimen. Incubate at 37°C: paraffin

sections for 30 minutes, cytological and frozen specimens for 10 minutes. Tap off proteolytic work

solution and dehydrate the slides in graded ethanol series (70%, 95% and 100%). Duration of each

soak is 1 minute. Air dry the slides and start with the hybridisation procedure. Do not treat more than

5 slides at the same time, because the temperature of the hot plate may drop dramatically, thus

causing incomplete proteolysis.

CHAPTER 2 – REMBRANDT® DETECTION PROTOCOL

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2.3 Hybridisation procedure

Denaturation and Hybridisation

Homogenize probe solutions. Apply 1 drop or 20 µl of probe solution (yellow/purple) to each

specimen and the positive control specimen. Apply 1 drop or 20 µl of the negative control probe

(green) to each negative procedure control specimen and apply 1 drop or 20 µl of the positive control

probe (pink) to each positive procedure control specimen. Cover all specimens with a cover slip

(avoid air bubbles!). Place slides on a 95°C hotplate and incubate for 5 minutes (denaturation). Work

in a preset order to ensure that all slides have been incubated at 95°C for the exact same time! Do not

denature more than 5 slides at the same time, because the temperature of the hot plate may drop

dramatically, thus causing incomplete denaturation. Transfer slides into a moist environment and

incubate for 16 hours at 37°C (during the hybridisation the minimum temperature should be room

temperature and the maximum temperature should be 37°C). Best results are obtained with prolonged

incubation time (16 hours).

Differentiation (stringency wash) and rinsing

− Remove coverslips by submerging the slides in TBS buffer. Soak the slides until the coverslips fall

off. Rinse the slides in TBS buffer for 10 minutes. Take the slides out, wipe off excess buffer and

dry the edges using a lint-free cloth. Please mind NOT to perform the differentiation step on

specimens incubated with the positive procedure control oligo probe (pink)!

− Apply 5-6 drops of the appropriate PanWash solution (white) to each specimen (except for the

positive procedure control) and transfer the slides onto a 37°C heating block or slide warmer.

Incubate for 15 minutes at 37°C. Rinse all slides 3x 1 minute in TBS buffer. Wipe off excess

reagent and start with the detection and staining procedure.

2.4 Detection and staining procedure

Apply 2-3 drops of AP-conjugate (red) to each specimen and transfer slides onto a 37°C heating

block or slide warmer. Incubate for 30 minutes at 37°C. Tap off excess detection reagent and rinse

the slides in TBS buffer. Soak 3x 1 minute in TBS buffer, while occasionally shaking the container.

Transfer the slides into a container with distilled or deionised water and soak slides for 1 additional

minute.

Take the slides out, wipe off excess of water and dry around the edges using a lint-free cloth. Ensure

that the specimen on the slide is not disrupted. Apply 2-3 drops of NBT/BCIP substrate (blue) to each

specimen and transfer the slides onto a 37°C heating block or slide warmer. Incubate in the dark for

5-15 minutes at 37°C (examine the colour development every 5 minutes microscopically). Tap off

excess substrate solution and rinse the slides for 3x 1 minute in changes of distilled or deionised

water. The slides are now ready to be mounted or counterstained.

2.5 Counterstain procedure

When a contrast colour is desired, the slides can be counterstained using Nuclear Fast Red (orange).

Wipe off excess reagent and apply 2-3 drops of counterstain to each specimen. Incubate for 1 minute

(longer incubation is possible and will yield stronger staining). Tap off excess counterstain and rinse

the slides briefly in distilled or deionised water. Mount the slides by using an aqueous mounting

medium. Interpret the results under the microscope.

CHAPTER 3 – LIMITATIONS OF PROCEDURE

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Chapter 3 Limitations of Procedure

3.1 Limitations

• The Rembrandt DNA and RNA in situ Hybridisation and Detection kits are solely applicable for

the detection of corresponding DNA or RNA which may be present in cell preparations (paraffin

sections, frozen sections or cytological specimen).

• Appropriate medical decisions are only possible if the medical traceability is ensured. The

product is intended for professional use as an aid in the diagnosis corresponding to the DNA or

RNA probes as supplied with the kit.

• Either human tissue sections or human cytological preparations may be used. Samples must be

fixed in buffered formalin or alcohol. Sections should be cut at 4 um thickness, glued to the

glass slides with a bio-adhesive (e.g. organosilane), dried at room temperature, subsequently

dried at 37 °C overnight, complete deparaffinisation in xylene and alcohol series and air dried.

Cytological specimen should be prepared as required by the user, fixed with cytological fixation

agent, rinsed in distilled water prior to the ISH procedure and air dried.

• Many factors can influence the performance of the ISH procedure. Failure in detection can be

due to i.e. improper sampling, handling, the time lapse between tissue removal and fixation, the

size of the tissue specimen in the fixation medium, the fixation time, processing fixed tissue, the

thickness of the section, the bio-adhesive on the slide, deparaffinisation procedure, incubation

times, proteolytic pre-treatment, detection reagents, incubation temperatures and interpretation

of results.

• The performance of the ISH procedure is also affected by the sensitivity of the method and the

DNA or RNA target load; in case the limit of the sensitivity is reached or when the target DNA

or RNA load is too low, a false negative reaction may be the result.

• The clinical interpretation of the results should not be established on the basis of a single test

result. A precise diagnosis, in fact, should take into consideration clinical history, symptoms, as

well as morphological data. Negative results therefore do not rule out any possibility of a

positive specimen.

• The Rembrandt test results are not to be relied on in case the sampling, sampling method,

quality, sample preparation, reagents used, controls and procedure followed is not optimal.

• Therapeutic considerations based on the result of this test alone should not been taken. Positive

results should be verified by other traditional diagnostic methods such as but not limited to

clinical history, symptoms, as well as morphological data.

• The medical profession should be aware of risks and factors influencing the intensity, the

absence or presence of probe signals which can not be foreseen when applying this test.

• The user should carefully consider the risk and use of sample material for this test in case the

sample material does not contain sufficient or representative test material.

• Laboratory personnel performing the test should be knowledgeable and be able to interpret the

test results.

3.2 Interpretation of the results

First, check the negative and positive controls that have been incubated with the test slides

simultaneously:

- The negative control should be really negative, i.e. not show any localised colour precipitations. If

the negative control could be interpreted as being positive, discard the results since no conclusions

can be drawn.

CHAPTER 3 – LIMITATIONS OF PROCEDURE

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- The positive control should show colour precipitations in conformity with the localisation of the

target DNA or RNA. The colour should show the proper shade and must be clearly visible in the

preferential cell/ tissue type and correspond to the target localisation.

In the test slides, start under low power magnification and focus on localisation and colour to see

whether:

- The positivity (colour precipitation) observed is localised in the cell type preferred by the target.

- The colour has the right shade (no endogenous or formalin pigment).

Use high power magnification to see whether:

- The positive staining texture (granular, etc), demarcation and localisation are conform the positive

control staining pattern.

Product in combination with other devices

The Rembrandt in situ Hybridisation and Detection kits are intended for stand-alone usage. The in

vitro diagnostic is intended to be used in combination with standard formalin fixed, paraffin

embedded tissue blocks, standard tissue freezing, tissue sectioning (microtome), standard cytological

preparation methods, hot plate(s), stove(s), incubation device(s), water bath(s), temperature and

incubation time control(s), and other reagents (not supplied with this reagent) and a microscope. The

combination has been tested and validated. Since the standard formalin fixed, paraffin embedded

tissue blocks, standard tissue freezing, tissue sectioning (microtome), standard cytological

preparation methods, hot plate(s), stove(s), incubation device(s), water bath(s), temperature controls,

incubation time control(s) and other not supplied reagents such as but not limited to fixation,

embedding and dewaxing reagents and a microscope is not combined with the device as a product,

conformity with the essential requirements is not applicable. Assay validation criteria are mentioned

in ‘Interpretation of the Results’ and are also depending on the target load, which may influence the

validation criteria.

Specifications of the DNA probes:

HPV1 Positive Control DNA Negative Control DNA

Specificity 95% 100% 100%

Sensitivity 85% 95% 95%

1 The specificity of the HPV DNA probes is 100% for the HPV species, but the different specific HPV probe sub-types may

show some inter-type cross reactions.

CHAPTER 4 – REFERENCES

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Chapter 4 References

1. Autillo-Touati A. et al., HPV Typing by In Situ Hybridization on Cervical Cytologic Smears with

ASCUS, Acta Cytologica, Vol. 42, p. 631-638, 1998.

2. Benkemoun A. et al., Evaluation of KREATECH In Situ Hybridization Kits for Detection of Human

Papillomavirus DNA on Cervical Smears with “ASCUS”, 3rd International Symposium “Impact of

Cancer Biotechnology Diagnostic & Prognostic Indicators”, Nice, France, October 1996. Accepted

for publication in Cancer Detection and Prevention.

3. Botma H.J. et al., Differential In Situ Hybridization for Herpes Simplex Virus Typing in Routine Skin

Biopsies, Journal of Virological Methods, Vol. 53, p. 37-45, 1995

4. Cooper K. et al., Human Papillomavirus DNA in Oesophageal Carcinomas in South Africa, Journal of

Pathology, Vol. 175, p. 273-277, 1995.

5. Davidson B. et al., Angiogenesis in Uterine Cervical Intraepithelial Neoplasia and Squamous Cell

Carcinoma: An Immunohistochemical Study, International Journal of Gynecological Pathology, Vol.

16, p. 335-338, 1997.

6. Davidson B. et al., CD44 Expression in Uterine Cervical Intraepithelial Neoplasia and Squamous Cell

Carcinoma: An Immunohistochemical Study, European Journal of Gyneacology and Oncology, Vol.

XIX, no. 1, p. 46-49, 1998.

7. Davidson B. et al., Inflammatory Response in Cervical Intraepithelial Neoplasia and Squamous Cell

Carcinoma of the Uterine Cervix, Pathology Research and Practice, Vol. 193, p. 491-495, 1997.

8. Gómez F. et al., Diagnosis of Genital Infection Caused by Human Papillomavirus Using In Situ

Hybridization: The Importance of the Size of the Biopsy Specimen, Journal of Clinical Pathology, Vol.

48, p. 57-58, 1995.

9. Jing X. et al., Detection of Epstein-Barr Virus DNA in Gastric Carcinoma with Lymphoid Stroma,

Viral Immunology, Vol. 10, No. 1, p. 49-58, 1997.

10. Sugawara I. et al., Detection of a Helicobacter Pylori Gene Marker in Gastric Biopsy Samples by

Non-Radioactive In Situ Hybridization, Acta Histochemica et Cytochemica, Vol. 28, No. 3, p. 263-267,

1995.

11. Van den Brink W. et al., Combined ß-Galactosidase and Immunogold/Silver Staining for

Immunohistochemistry and DNA In Situ Hybridization, Journal of Histochemistry and Cytochemistry,

Vol. 38, p. 325-329, 1990.

12. Yanai H. et al., Epstein-Barr Virus Infection in Non-Carcinomatous Gastric Epithelium, Journal of

Pathology, Vol. 183, p. 293-298, 1997.

13. Yonezawa S. et al., MUC2 Gene Expression is Found in Non-invasive Tumors But Not in Invasive

Tumors of the Pancreas and Liver: Its Close Relationship with Prognosis of the Patients, Human

Pathology, Vol. 28, No. 3, p. 344-352, 1997.

14. Ziol M. et al., Virological and Biological Characteristics of Cervical Intraepithelial Neoplasia grade I

with marked koilocytic Atypia, Human pathology, Vol 29, No. 10, p. 1068-1073, 1998.

15. Evans M. et al, Biotinyl-Tyramide-Based In Situ Hybridization Signal Patterns Distinguish Human

Paplliloma Virus Type and Grade of Cervical Intraepithelial Neoplasia, Mod Pathol 2002;

15(12):1339-1347

16. Hopman A. et al., Transition of high grade cervical intraepithelial neoplasia to micro-invasive

carcinoma is characterized by integration of HPV 16/18 and numerical chromosome abnormalities, J

Pathol 2004; 202:23-33

17. Hopman A. et al., Human papillomavirus integration: detection by in situ hybridization and potential

clinical application, J Pathol 2004; 202:1-4

18. Hafkamp et al., A subset of head and neck squamous cell carcinomas exhibits integration of HPV

16/18 DNA and overexpression of p16INK4A and p53 in the absence of mutations in p53 exons 5-8, Int.

J. Cancer; 107(3):394-400

CHAPTER 5 – PROBE SPECIFICATIONS

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Chapter 5 Probe specifications

REMBRANDT® Biotin and Digoxigenin1 labelled DNA probe specifications

PRODUCT

CODE

LABEL

DNA PROBE SPECIFICATIONS

Description

Size

Region

Vector

A100P.0100

A100P.9900

BIO

DIG

Human papilloma virus HPV screening

DNA probe (PROBE xxx panHPV)*

100-300 bp mix of total genomes 7-8 Kb; containing

the conserved HPV region

pBR322; 4.3 Kb and pSP;

3.0 Kb

A191P.0100

A191P.9900

BIO

DIG

Human papilloma virus type 6/11 DNA

probe (PROBE xxx HPV 6/11)*

100-300 bp total genome 7.8 Kb HPV type 6 and 7.9

Kb HPV type 11

pSP; 3.0 Kb

A192P.0100

A192P.9900

BIO

DIG

Human papilloma virus type 16/18 DNA

probe (PROBE xxx HPV 16/18)*

100-300 bp total genome 7.9 Kb HPV type 16 and 7.9

Kb HPV type 18

pSP; 3.0 Kb

A193P.0100

A193P.9900

BIO

DIG

Human papilloma virus type 31/33 DNA

probe (PROBE xxx HPV 31/33)*

100-300 bp total genome 7.9 Kb HPV type 31 and 7.9

Kb HPV type 33

pBR322; 4.3 Kb and

modified pSP ≈ 4.0 Kb

A106P.0100

A106P.9900

BIO

DIG

Human papilloma virus type 6 DNA

probe (PROBE xxx HPV 6)*

100-300 bp total genome 7.8 Kb HPV type 6 pSP; 3.0 Kb

A111P.0100

A111P.9900

BIO

DIG

Human papilloma virus type 11 DNA

probe (PROBE xxx HPV 11)*

100-300 bp total genome 7.9 Kb HPV type 11 pSP; 3.0 Kb

A116P.0100

A116P.9900

BIO

DIG

Human papilloma virus type 16 DNA

probe (PROBE xxx HPV 16)*

100-300 bp total genome 7.9 Kb HPV type 16 pSP; 3.0 Kb

A118P.0100

A118P.9900

BIO

DIG

Human papilloma virus type 18 DNA

probe (PROBE xxx HPV 18)*

total genome 7.9 Kb HPV type 18 pSP; 3.0 Kb

A131P.0100

A131P.9900

BIO

DIG

Human papilloma virus type 31 DNA

probe (PROBE xxx HPV 31)*

100-300 bp total genome 7.9 Kb HPV type 31 pBR322; 4.3 Kb

A133P.0100

A133P.9900

BIO

DIG

Human papilloma virus type 33 DNA

probe (PROBE xxx HPV 33)*

100-300 bp total genome 7.9 Kb HPV type 33 modified pSP ≈ 4.0 Kb

Q001P.0100

Q001P.9900

BIO

DIG

Negative control probe for DNA

(CONTROL – xxx DISH)*

100-300 bp pSP; 3.0 Kb

Q151P.0100

Q151P.9900

BIO

DIG

Positive control probe for DNA

(CONTROL + xxx DISH)*

30-mer

oligonucleotide

Mixture of six oligonucleotides

complimentary to ALU repeats

* xxx = label (BIO or DIG)

1 Digoxigenin (DIG) labeling and detection is protected by international patents of Roche Molecular Biochemicals. This product is supplied under a license of Roche Molecular

Biochemicals. This product or the use of this product may be covered by one or more patents of Roche Molecular Biochemicals, including the following: EP patent 0324 474

(granted); U.S. patent 5.354.657 (granted).

CHAPTER 5 – PROBE SPECIFICATIONS

PANPATH REMBRANDT ® DETECTION KITS PAGE 12 OF 20

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Contents : - clear vial, yellow cap = BIO labelled probe; 1 mL (10-100 assays)

- clear vial, purple cap = DIG labelled probe; 1 mL (10-100 assays)

- white vial, white cap = matching PanWash; 15 mL

R013R.0000 for probes with GC % < 50%

R014R.0000 for probes with GC % > 50%

Format : ready to use

Application : colorimetric detection of respective DNA in human specimen by in situ hybridisation (ISH)

Purification : by size exclusion chromatography

Detection limit : 3-10 pg by filter hybridisation

Storage : refrigerated (2-8 °C); do not freeze

Stability : until expiry date printed on label

Precautions : - homogenise solutions before use

- avoid contact with eyes and skin; do not swallow

CHAPTER 6 – TROUBLE SHOOTI NG GUIDE

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Chapter 6 Trouble Shooting Guide

6.1 Introduction

This Trouble Shooting Guide is intended to support you in obtaining optimal results with PanPath's

REMBRANDT® In Situ Hybridisation and Detection kits.

In the next pages we inform you not only about possible causes and remedies for often occurring

problems when performing ISH, but we also provide you with some tips given by experts on In Situ

hybridisation that may be of help to you.

It is of course always possible that you encounter a problem which is not covered by this Trouble

Shooting Guide, or that you still have doubts about your results. In such cases, please do not hesitate

to contact your local supplier or PanPath directly. Since we consider your problem as our problem,

we will do our utmost to find a proper solution.

6.2 No section or cells left on the slides

Possible causes

Remedies

 Sample preparation.

→

Make sure samples are prepared according to

protocol, the tissue is fixed in neutral buffered

formalin and the slides are air dried well.

 Tissue section too thin.

→

Optimal thickness of the tissue is 4-6 µm.



Wrong (side of) glass slide used.

→

Use only organosilane coated glass slides.

 Pepsin conce ntration too high.

→

Make sure correct concentration of pepsin is used

(depending on type of specimen).

 Digestion step too long.

→

Reduce digestion time (15 minutes instead of 30

minutes) or digest at room temperature.



Denaturation.

→

Make sure temperature is 95 ± 5°C.

→ Denatu re no longer than 5 minutes.

 Coverslips removed with force.

→

Make sure that slides are soaked for at least 10

minutes in PBS.

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6.3 Weak or no staining on a suspected positive sample

Possible causes

Remedies

 Tissue fixation.

→

Only use buffered formalin fixative.



Deparaffinization.

→

Refresh dewax series.

 Digestion.

→

Make sure correct concentration of pepsin is used.

→ Make sure digestion takes place at 37°C.

 Denaturation.

→

Make sure temperature is 95 ± 5°C.

 Interfering internal structures of probes.

→

In case of RISH procedures, warm up probe solution at

85°C for 5 min. before usage.

 Hybridisation procedure.

→

Homogenize probe solution prior to applying probe on

the section.

 Washing temperature.

→

Make sure temperature is 37 ± 2°C.

→ Depending on GC%, make sure correct PanWash

(Differentiation reagent) is used.



Detection procedure.

→

Make sure temperature is 37 ± 2°C.

→ Make sure to incubate in the dark.



Low amount of target DNA.

→

Prolong hybridisation.

 Colour precipitate rinsed away

→

Make sure that proper rinse and mounting media are

used.

6.4 Negative staining of the positive control

Possible causes

Remedies

 Deparaffinization

→

Re-fresh dewax series.

 Positive control specimen incubated with

positive control probe washed with PanWash.

(Differentiation reagent)

→

Do not use PanWash (Differentiation reagent) on

positive control specimen.

 Denaturation temperature.

→

Make sure temperature is 95 ± 5°C.



Interfering internal structures of probes.

→

In case of RISH procedures, warm up probe

solution at 85°C for 5 min. before usage.



Detection procedure.

→

Make sure temperature is 37°C ± 2°C.

CHAPTER 6 – TROUBLE SHOOTI NG GUIDE

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6.5 Positive staining of the negative control

Possible causes

Remedies

 Drying out of the section.

→

Incubate in a moisturised environment.



Washing procedure.

→

Make sure temperature is 37 ± 2°C.

→ Depending on GC%, make sure correct

PanWash (Differentiation reagent) is used.

 Contamination with positive control probe or

specific probe.

→

Make sure that the positive control probe is the

latest to be applied to the section.

6.6 Non-specific background staining

One should always bear in mind that the staining intensity and the level of background (or non-

specific) staining may depend on the type of tissue used.

Possible causes

Remedies

 Tissue section too thick. → Optimal thickness of the tissue is 4-6 µm.

 Tissue crumbled. → Make sure tissue is stretched completely.

 Deparaffinization. → Dewax series

 Denaturation temperature too high. → Make sure temperature is 95 ± 5°C.

 Denaturation step too long. → Denature no longer tha n 5 minutes.

 Drying out of the section. → Incubate all procedure steps in a moisturised environment;

prevent evaporation

 Washing temperature. → Make sure temperature is 37 ± 2°C.

 Substrate incubation step too long. → Shorten incubation time with 5 minutes.

 Endogenous peroxidase. → Inactivate endogenous peroxidase by incubating tissue

sections in 3% H2O2/H2O for 15 minutes at room

temperature prior to the digestion step.

 Endogenous alkaline phosphatase. → Inactivate endogenous alkaline phosphatase by incubating

sections in substrate solution to which 4 mg of levamisole is

added.

6.7 Cross Hybridisation

One should always bear in mind that there is a possibility of cross hybridisation between related

subtypes and that a patient can be infected with more than one subtype of a virus.

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REFERENCE GUIDE-

DISH-AP

PRETREATMENT OF PARAFFIN SECTIONS

INCUBATION TIME

1. Cut 4-6 µm sections and collect on treated glass slides

2. Heat slides 2-16 hrs at 56-60°C

3. Dewax in fresh xylene 2 x 10 min.

4. Soak slides in 100% ethanol and air dry 5 min.

PROTEOLYTIC TREATMENT

1. Upon receipt of the kit dissolve pepsin powder in 8 mL distilled/ deionized water,

aliquot in 150 µL batches and freeze at -20°C.

2. Dilute the 1M HCl pepsin diluent (transparent) to the application required

concentration (paraffin, cytological or frozen; see 3)

3. Dilute thawed proteolytic stock solution in diluted HCl and incubate each specimen

with 300-400 µl:

paraffin: 100x in 0.1M HCl;

add 100 µl to 5 ml 0.1M HCl 30 min. 37°C heating block

cytological: 25,000x in 0.01M HCl;

add 8 µl to 100 ml 0.01M HCl 10 min. 37°C heating block

frozen: 50,000x in 0.01M HCl;

add 4 µl to 100 ml 0.01M HCl 10 min. 37°C heating block

4. Discard excess proteolytic work solution

5. Dehydrate slides in graded ethanol and air dry 3 x 1 min.

HYBRIDIZATION PROCEDURE

1. Apply 1 drop or 20 µl of probe solution per specimen; cover with coverslip

2. Denature 5 min. 95°C heating block

3. Hybridize 16 hrs 37°C incubator

4. Remove coverslips by soaking slides in TBS buffer 10 min.

5. Apply 5-6 drops of PanWash (white) to each specimen 15 min. 37°C heating block

except the positive control

6. Wash all slides in TBS buffer 3 x 1 min.

ETECTION AND STAINING PROCEDURE

1. Apply 2-3 drops of the conjugate (red) to each specimen 30 min. 37°C heating block

2. Soak slides in TBS buffer 3 x 1 min.

3. Soak slides in distilled/deionized water 1 min.

4. Apply 2-3 drops of NBT/BCIP substrate (blue) to each specimen

and incubate in dark 5-15 min. 37°C heating block

5. Tap off excess substrate solution and wash slides in distilled/deionized water 3 x 1 min.

6. Optional: apply 2-3 drops of counterstain (orange) to each specimen 1 min.

7. Wash slides in distilled/deionized water 3 x 1 min.

8. Mount sections for microscopic evaluation

VIAL-

DIGEST

PROBE

WASH

PANWASH

WASH

CONJ

WASH

SUBS

COUNT

LABEL

PEPSIN POW

PEPSIN DIL

……………

TBS

GC%.....50%

TBS

....... AP

TBS

NBT/BCIP

NFR

ANLEITUNG-

DISH-AP

ERSTELLUNG VON

ARAFFINSCHNITTEN

INKUBATIONSZEITEN

1. 4-6 µm Schnitte anfertigen und auf vorbehandelte Objektträger ziehen

2. Schnitte inkubieren 2-16 Stunden bei 56-60°C

3. in Xylol entparaffinieren 2 x 10 Min.

4. Schnitte in 100% Ethanol entwässern und lufttrocknen 5 Min.

PROTEOLYTISCHE

EHANDLUNG

1. Pepsin Pulver in 8 mL destilliertem/deionisiertem Wasser lösen, in 150 µL

Portionen aliquotieren und bei -20°C aufbewahren.

2. 1M HCl Pepsin-Lösungsmittel (Transparent) auf die Konzentration

verdünnen, die für die entsprechende Anwendung notwendig ist

(Paraffinschnitt, Zytologisches Präparat oder Gefrierschnitt; siehe 3)

3. Verdünne die aufgetaute proteolytische Lösung in verdünntem HCl; jedes

Präparat mit 300-400 µL inkubieren.

Paraffinschnitt: 100x in 0.1M HCl;

100 µL proteolytische Lösung zu 5 mL 0.1M HCl geben 30 Min. bei 37°C Heizplatte

Zytologisches Praeparat: 25,000x in 0.01M HCl;

8 µL proteolytische Lösung zu 100 mL 0.01M HCl geben 10 Min bei 37°C Heizplatte

Gefrierschnitt: 50,000x in 0.01M HCl;

4 µL proteolytische Lösung zu 100 mL 0.01M HCl geben 10 Min. bei 37°C Heizplatte

4. Überschüssige proteolytische Lösung verwerfen

5. Schnitte in Ethanolreihe entwässern und Lufttrocken 3 x 1 Min.

HYBRIDISIERUNGSPROZEDUR

1. 1 Tropfen oder 20 µl der Sonde auf jedes Präparat geben und mit einem

Deckglas abdecken

2. Denaturieren 5 Min. bei 95°C Heizplatte

3. Hybridisieren 16 Stunden bei 37°C Ofen

4. Entfernen der Deckgläser durch Eintauchen in TBS Puffer 10 Min.

5. 5-6 Tropfen PanWash (Weiss) zu jedem Präparat geben

(mit Ausnahme der Positivkontrolle) 15 Min. bei 37°C Heizplatte

6. Präparate in TBS Puffer spülen 3 x 1 Min.

ETEKTIONS

UND

ÄRBEPROZEDUR

1. 2-3 Tropfen Konjugat (Rot) auf jedes Präparat geben 30 Min. bei 37°C Heizplatte

2. Präparate in TBS Puffer spülen 3 x 1 Min.

3. Präparate mit destilliertem/deionisiertem Wasser spülen 1 Min.

4. 2-3 Tropfen des NBT/BCIP-Substrats (Blau) auf jedes Präparat geben

Im dunkeln inkubieren 5-15 Min. bei 37°C Heizplatte

5. In destilliertem/deionisiertem Wasser spülen 3 x 1 Min.

6. Optional: 2-3 Tropfen der Gegenfärbelösung (Orange) auf jedes Präparat geben

7. In destilliertem/deionisiertem Wasser spülen 3 x 1 Min.

8. Präparate für die mikroskopische Beurteilung abdecken

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GUIDE REFERENCE-

DISH-AP

PRE

TRAITEMENT DES SECTIONS PARAFFINEES

TEMPS D

’

INCUBATION

1. Préparez des sections de 4-6 µm et coller les en lames traitées

2. Chauffez les lames 2-16 hrs à 56-60°C

3. Deparaffinez dans du xylène frais 2 x 10 min.

4. Immergez dans de l’éthanol 100% (ou ethanol absolu) et laissez

séchez à l’air 5 min.

TRAITEMENT PROTEOLYTIQUE

1. Dissolvez la poudre de pepsine dans 8 ml d’eau distillée ou deinonisée,

divisez la solution en aliquotes de 150 µl et congelez les (-20°C).

2. Diluez la solution HCl 1M (solution de pepsine en tant que diluant;

transparent) selon l’usage préféré (paraffiné, cytologique ou congelé)

3. Diluez une aliquote de la solution du stock proteolytique avec la solution diluée de

HCl et incubez chaque échantillon dans 300-400 µl de la manière suivante:

dilution paraffinée: 100x dans 0.1M HCl ;

ajouter 100 µl à 5 ml de HCl 0.01M 30 min. 37°C bloc chauffant

dilution cytologique: 25,000x dans 0.01M HCl ;

ajouter 8 µl à 100 ml HCl 0.01M 10 min. 37°C bloc chauffant

dilution congelée: 50,000x dans 0.01M HCl ;

ajouter 4 µl à 100 ml de HCl 0.01M 10 min. 37°C bloc chauffant

4. Rincez avec de l’eau distillée ou deionisée

5. Deshydratez les lames dans une série d’éthanol et laissez sécher à l’air 3 x 1 min.

ROTOCOLE D

’

HYBRIDATION

1. Ajoutez une goutte ou 20 µl d’une solution de sonde par échantillon et

couvrez avec une lamelle

2. Dénaturez 5 min. 95°C bloc chauffant

3. Hybridez 16 hrs 37°C incubateur

4. Diffusez verticalement le tampon TBS sur les lamelles 10 min.

5. Ajoutez 5-6 gouttes de PanWash (blanc) à chaque échantillon 15 min. 37°C bloc chauffant

à l’exception du contrôle positif

6. Rincez toutes les lames avec le tampon TBS 3 x 1 min.

ROTOCOLE DE

ETECTION ET DE COLORATION

1. Distribuez 2-3 gouttes de conjugant (rouge) par spécimen 30 min. 37°C bloc chauffant

2. Immergez les lames dans le tampon TBS 3 x 1 min.

3. Immergez les lames dans de l’eau distillée ou deionisée 1 min.

4. Ajoutez 2-3 gouttes du substrat NBT/BCIP (bleu) à chaque échantillon

et incubez dans l’obscurité 5-15 min. 37°C bloc chauffan

5. Rincez les lames avec de l’eau distillée ou deionisée 3 x 1 min.

6. Optionnel: ajoutez 2-3 gouttes de la solution colorante (orange) 1 min.

7. Rincez les lames avec de l’eau distillée ou deionisée 3 x 1 min.

8. Montez les sections et examinez les lames au microscope

VIAL-

DIGEST

PROBE

WASH

PANWASH

WASH

CONJ

WASH

SUBS

COUNT

LABEL

PEPSIN POW

PEPSIN DIL

……………

TBS

GC%.....50%

TBS

....... AP

TBS

NBT/BCIP

NFR

GUIA DE REFERENCIA-

DISH-AP

RETRATAMIENTO DE LOS CORTES DE

ARAFINA

IEMPO DE INCUBATION

1. Preparar cortes de 4-6 µm y depositarlas sobre portas tratados

2. Calentar los portas 2-16 horas A 56-60ºC

3. Desparafinar en xileno límpio 2 x 10 minutos

4. Sumergir los portas en etanol absoluto y secar al aire 5 minutos

RATTAMIENTO

ROTEOLITICO

1. A la recepción del kit disolver la pepsina en polvo en 8 ml de agua

destilada/desionizada, hacer alícuotas de 150 µl y congelar a –20ºC.

2. Diluir el diluyente de la pepsina (vial transparente) ClH 1M a la

concentración requerida para la aplicación (parafina, citología o

congelación; ver 3)

3. Diluir la solución proteolítica stock descongelada en ClH diluido e incubar

cada muestra con 300-400 µl:

Parafina: 100X en ClH 0.1M;

añadir 100 µl a 5 ml de ClH 0.1M 30 minutos en un termobloque a 37ºC.

Citología: 25.000X en ClH 0.01M ;

añadir 8 µl a 100ml de ClH 0.01M 10 minutos en un termobloque a 37ºC

Congelación: 50.000X en ClH 0.01M;

añadir 4 µl a 100 ml de ClH 0.01M 10 minutos en un termobloque a 37ºC

4. Eliminar el exceso de solución proteolítica a la dilución de trabajo

5. Deshidratar los portas en soluciones alcohólicas crecienbtes y secar al aire 3 x 1 minuto

ROTOCOLO DE HIBRIDACION

1. Añadir 1 gota o 20 µl de la solución de la sonda por muestra.

Cubrir con un cubre.

2. Desnaturalizar 5 min. a 95ºC en una placa calefactora

3. Hibridizar 16 horas a 37ºC en un incubador

4. Retirar los cubres sumergiendo los portas en tampón TBS 10 minutos

5. Añadir 5-6 gotas de Pan Wash (vial blanco) a cada muestra 15 minutos en un termobloque a 37ºC.

excepto al control positivo

6. Lavar todos los portas en tampón TBS 3 x 1 minuto

ROTOCOLO DE DETECCION Y TINCION

1. Añadir 2-3 gotas del conjugado (vial rojo) a cada muestra 30 minutos a 37ºC en

un termobloque

2. Sumergir los portas en tampón TBS 3 x 1 minuto

3. Sumegir los portas en agua desionizada 1 minuto

4. Añadir 2-3 gotas del substrato NBT/BCIP (vial azul) a cada muestra e 5-15 minutos a 37ºC en un termobloque

incubar en oscuridad

5. Eliminar el exceso de solución de substrato y lavar los portas en agua 3 x 1 minuto

destilada o desionizada

6. Opcionalmente: añadir 2-3 gotas de solución de contraste (vial naranja) a 1 minuto

cada muestra

7. Lavar los portas en agua desionizada 3 x 1 minuto

8. Montar los portas para su evaluación por microscopía

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ANDLEIDING

DISH-AP

NIJDEN EN PLAKKEN VAN PARAFFINE COUPES

INCUBATIE TIJDEN

1. Snij 4-6 µm coupes en plak ze op voorbehandelde objectglaasjes

2. Verwarm de glaasjes 2-16 uur bij 56-60°C

3. Deparaffineer in verse xylol 2 x 10 min.

4. Spoel in 100% ethanol en laat de glaasjes luchtdrogen 5 min.

PROTEOLYTISCHE VOORBEHANDELING

1. Los het pepsine poeder in 8 mL gedestilleerd/gedeioniseerd water op,

verdeel in 150 µL porties en bewaar bij -20°C.

2. Verdun het 1M HCl pepsine oplosmiddel (transparant) tot de concentratie

die voor de applicatie nodig is (paraffine, cytologie of vries coupe; zie 3)

3. Verdun de proteolytische stock oplossing in het verdunde HCl

en incubeer elk preparaat met 300-400 µL:

paraffine: 100x in 0.1M HCl;

voeg 100 µL aan 5 mL 0.1M HCl toe 30 min. bij 37°C hete plaat

cytologie:25,000x in 0.01M HCl;

voeg 8 µL an 100 mL 0.01M HCl toe 10 min. bij 37°C hete plaat

vries: 50,000x in 0.01M HCl;

voeg 4 µL aan 100 mL 0.01M HCl toe 10 min. bij 37°C hete plaat

4. Overmaat van de proteolytische werkoplossing weggooien (altijd vers bereiden)

5. Ontwater de preparaten in oplopende alcoholreeks en luchtdrogen 3 x 1 min.

HYBRIDISATIE PROCEDURE

1. Incubeer elk preparaat met probe reagens; 1 druppel of 20 µl

en dek af met dekglaasje

2. Denatureer 5 min. bij 95°C hete plaat

3. Hybridiseer 16 uur bij 37°C stoof

4. Verwijder dekglaasje door preparaten in TBS buffer te dompelen 10 min.

5. Incubeer elk preparaat met PanWash (wit); 5-6 druppels

met uitzondering van de positieve controle 15 min. bij 37°C hete plaat

6. Spoel alle preparaten in TBS buffer 3 x 1 min.

ETECTIE EN TEGENKLEURINGSPROCEDURE

1. Incubeer elk preparaat met conjugaat (rood); 2-3 druppels 30 min. bij 37°C hete plaat

2. Spoel de preparaten met TBS buffer 3 x 1 min.

3. Spoel de preparaten met gedestilleerd/gedeioniseerd water 1 min.

4. Incubeer elk preparaat met NBT/BCIP substraat (blauw); 2-3 druppels

en incubeer in donker 5-15 min. bij 37°C hete plaat

5. Spoel de preparaten met gedestilleerd/gedeioniseerd water 3 x 1 min.

6. Optioneel: incubeer elk preparaat met tegenkleuring (oranje); 2-3 druppels 1 min.

7. Spoel de preparaten met gedestilleerd/gedeioniseerd water 3 x 1 min.

8. Dek de coupes af

VIAL-

DIGEST

PROBE

WASH

PANWASH

WASH

CONJ

WASH

SUBS

COUNT

LABEL

PEPSIN POW

PEPSIN DIL

……………

TBS

GC%.....50%

TBS

....... AP

TBS

NBT/BCIP

NFR

ETODICA D

’U

DISH-AP

PRETRATTAMENTO DELLE SEZIONI IN PARAFFINA

TEMPI DI INCUBAZIONE

1. Tagliare sezioni di 4-6 µm e depositarele su vetrino trattato

2. Scaldare i vetrini 2-16 ore a 56-60°C

3. Togliere la paraffina usando xilolo fresco 2 x 10 min.

4. Sciacquare i vetrini in etanolo 100% ed asciugare all’aria 5 min.

TRATTAMENTO PROTEOLITICO

1. Seguendo le istruzioni del kit, sciogliere la pepsina in polvere in 8 mL

di acqua distillata/deionizzata, quindi preparare aliquote di 150 µL e

congelare a -20°C.

2. Diluire la pepsina 1M HCl (trasparente) diluente sino a raggiungere la

concentrazione richiesta dall’applicazione (Paraffinati, Citologici o

Congelati; vedasi punto 3)

3. Diluire la soluzione proteolitica scongelata in HCl diluito secondo lo

schema seguente (Incubare ogni sezione con 300-400 µL):

Paraffinati: 100x in 0.1M HCl;

aggiungere 100 µL a 5 mL 0.1M HCl 30 min. su blocco riscaldante a 37°C

Citologici: 25’000x in 0.01M HCl;

aggiungere 8 µL a 100 mL 0.01M HCl 10 min. su blocco riscaldante a 37°C

Congelati: 50’000x in 0.01M HCl;

aggiungere 4 µL a 100 mL 0.01M HCl 10 min. su blocco riscaldante a 37°C

4. Gettare l’eccedenza della soluzione proteolitica

5. Disidratare le sezioni in etanolo puro ed asciugare all’aria 3 x 1 min.

PROCEDIMENTO DI IBRIDAZIONE

1. Aggiungere 1 goccia o 20 µl di soluzione “probe” su ogni sezione.

Coprire con coprivetrino.

2. Denaturare 5 min. su blocco riscaldante a 95°C

3. Ibridizzare 16 ore a 37°C in incubatrice

4. Togliere il coprivetrino sciacquando il vetrino in tampone TBS 10 min.

5. Aggiungere 5-6 gocce di PanWash (bianco) su ogni sezione

tranne sul controllo positivo 15 min. su blocco riscaldante a 37°C

6. Lavare tutti i vetrini in tampone TBS 3 x 1 min.

PROCEDIMENTO DI DETEZIONE E COLORAZIONE

1. Aggiungere ad ogni sezione 2-3 gocce di coniugato (rosso) 30 min. su blocco riscaldante a 37°C

2. Sciacquare i vetrini in tampone TBS 3 x 1 min.

3. Sciacquare i vetrini in acqua distillata/deionizzata 1 min.

4. Aggiungere ad ogni sezione 2-3 gocce di substrato NBT/BCIP (blu)

e incubare al buio 5-15 min. su blocco riscaldante a 37°C

5. Togliere l’eccesso di soluzione substrato e lavare i vetrini in

acqua distillata/deionizzata 3 x 1 min.

6. Opzionale: aggiungere ad ogni sezione 2-3 gocce di “counterstain”

(arancione) 1 min.

7. Lavare le sezioni in acqua distillata/deionizzata 3 x 1 min.

8. Preparare le sezioni per l’osservazione al microscopio

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PANPATH REMBRANDT ® DETECTION KITS PAGE 19 OF 20

PanPath B.V. Instraat 5b3 - NL6021 AC Budel Tel.: +31 (0)495 499090 - info@panpath.nl - www.panpath.nl

Οδηγός Αναφοράς-

DISH-AP

ΠΡΟΕΤΟΙΜΑΣΙΑ ΤΟΜΩΝ ΠΑΡΑΦΙΝΗΣ ΧΡΟΝΟΣ ΕΠΩΑΣΗΣ

1. Κόβετε τομές πάχους 4-6 μm και επιστρώνετε

σε επεξεργασμένες γυάλινες αντικειμενοφόρους πλάκες.

2. Επωάζετε σε κλίβανο 2-16 ώρες στους 56-60°C

3. Αποπαραφινώνετε σε φρέσκια ξυλόλη 2x10 min.

4. Εμβαπτίζετε τις τομές σε 100% αιθανόλη και στεγνώνετε 5 min.

ΠΡΩΤΕΟΛΥΤΙΚΗ ΠΕΨΗ

1. Άμεσα μετά την παραλαβή του κιτ, διαλύετε την πεψίνη σε 8 mL

αποστειρωμένο / απιονισμένο νερό, διαχωρίζετε σε κλάσματα των 150 μL

και καταψύχετε στους -20°C

2. Αραιώνετε το διαυγές διάλυμα της πεψίνης σε 1M HCl, σύμφωνα με την

επιθυμητή συγκέντρωση για τις εφαρμογές (σε παραφίνη, κυτταρολογικά

ή κατεψυγμένα δείγματα, σύμφωνα με το επόμενο βήμα 3)

3. Αραιώνετε το αποψυγμένο αποθεματικό διάλυμα του πρωτεολυτικού

ενζύμου σε αραιωμένο HCl και επωάστε κάθε δείγμα με 300-400 μL :

δείγματα σε παραφίνη: 100x σε 0,1M HCl;

Προσθέστε 100 μL σε 5 mL 0,1M HCl 30 min. σε θερμαινόμενη εστία 37°C

κυτταρολογικά δείγματα: 25.000x σε 0,01M HCl;

Προσθέστε 8 μL σε 100 mL 0,01M HCl 10 min. σε θερμαινόμενη εστία 37°C

κατεψυγμένα δείγματα: 50.000x σε 0,01M HCl;

Προσθέστε 4 μL σε 100 mL 0,01M HCl 10 min. σε θερμαινόμενη εστία 37°C

4. Απορρίπτετε το πλεονάζον διάλυμα πρωτεολυτικού ενζύμου

5. Πραγματοποιείτε αφυδάτωση σε κατιούσα αλκοολών και στεγνώνετε τις τομές 3x1 min.

ΔΙΑΔΙΚΑΣΙΑ ΥΒΡΙΔΟΠΟΙΗΣΗΣ

1. Προσθέστε 1 σταγόνα ή 20 μl από το διάλυμα του ανιχνευτή ανά δείγμα.

Σκεπάστε με καλυπτρίδα

2. Αποδιατάξτε 5 min σε θερμική πλάκα 95°C

3. Υβριδοποιήστε 16 ώρες σε επωαστικό θάλαμο 37°C

4. Αφαιρέστε τις καλυπτρίδες εμβαπτίζοντας τα πλακίδια σε διάλυμα TBS 10 min.

5. Προσθέστε 5-6 σταγόνες PanWash (λευκό)

σε κάθε δείγμα, εκτός του θετικού μάρτυρα 15 min σε θερμαινόμενη εστία 37°C

6. Έκπλυση όλων των πλακιδίων σε διάλυμα TBS 3x1 min.

ΔΙΑΔΙΚΑΣΙΕΣ ΑΝΙΧΝΕΥΣΗΣ ΚΑΙ ΧΡΩΣΗΣ

1. Προσθέστε 2-3 σταγόνες αντιδραστηρίου σύζευξης

(κόκκινο) σε κάθε δείγμα 30 min σε θερμαινόμενη εστία 37°C

2. Εμβαπτίστε τα πλακίδια σε διάλυμα TBS 3x1 min.

3. Εμβαπτίστε τα πλακίδια σε απιονισμένο νερό 1 min.

4. Προσθέστε 2-3 σταγόνες υποστρώματος NBT/BCIP

(μπλε) σε κάθε δείγμα και επωάστε σε σκοτάδι 5-15 min σε θερμαινόμενη εστία 37°C

5. Απορρίψτε το πλεονάζον διάλυμα υποστρώματος και ξεπλύνετε

τα πλακίδια με αποστειρωμένο ή απιονισμένο νερό 3x1 min.

6. Προαιρετικά: προσθέστε 2-3 σταγόνες διαλύματος

αντίχρωσης (πορτοκαλί) σε κάθε δείγμα 1 min.

7. Ξεπλύνετε τα πλακίδια σε απιονισμένο νερό 3x1 min.

8. Επιστρώστε τις τομές για μικροσκοπικό έλεγχο

VIAL-

DIGEST

PROBE

WASH

PANWASH

WASH

CONJ

WASH

SUBS

COUNT

LABEL

PEPSIN POW

PEPSIN DIL

……………

TBS

GC%.....50%

TBS

....... AP

TBS

NBT/BCIP

NFR

KIT MANUAL REMBRANDT® DIS H & AP DETECTION

PANPATH REMBRANDT ® DETECTION KITS PAGE 20 OF 20

PanPath B.V. Instraat 5b3 - NL6021 AC Budel Tel.: +31 (0)495 499090 - info@panpath.nl - www.panpath.nl

Immaterial property information

Digoxigenin (DIG) labeling and detection is protected by international patents of Roche Molecular

Biochemicals. This product is sold under a license of Roche Molecular Biochemicals. This product or the use

of this product may be covered by one or more patents of Roche Molecular Biochemicals, including the

following: EP 0324474, US 5,354,657.

REMBRANDT® is a registered trade name of PanPath BV, Budel, The Netherlands.

Purchase does not include the right to exploit this product commercially and any commercial use without the

explicit authorization of PanPath BV is prohibited.

Thermo Fisher Scientific REMBRANDT DISH and AP Mode d'emploi

Marque: Thermo Fisher Scientific

Modèle: REMBRANDT DISH and AP

Taper: Mode d'emploi

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English: Thermo Fisher Scientific REMBRANDT DISH and AP User guide

Thermo Fisher Scientific REMBRANDT DISH and AP Mode d'emploi

Thermo Fisher Scientific REMBRANDT DISH and AP Mode d'emploi

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