Protocol - Immunohistochemistry on sections
Immunohistochemistry on paraffin embedded sections
Category:
In situ hybridization and Immunochemistry
Last revision: Dec 10, 2014
Contact
Name: Giovanna L. Liguori
Address: Institute of Genetics and Biophysics "A. Buzzati-Traverso", CNR. Via Pietro Castellino 111, 80131 Napoli
Email: giovanna.liguori@igb.cnr.it
Figure legend:
Beta-catenin immunodetection on mouse colon tumor sections
Steps
| Description | Temperature | Time | Note |
|---|---|---|---|
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Put the slides in an apposite rack
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Wash slides in Xylene
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2x20min
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Check that paraffin disappears
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Wash slides in 100% EtOH (after Xylene)
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1x3min
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Sections become white
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Wash slides in 100% EtOH
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1x5min
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Wash slides in 95% EtOH
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1x5min
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Wash slides in 75% EtOH
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1x5min
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Wash slides in 50% EtOH
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1x5min
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Wash slides in PBS 1X
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2x5min
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Block endogenous peroxidase by incubating in MetOH-0.3% H2O2
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30min
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Wash slides in PBS 1X
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2x5min
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Put on the sections the blocking solutions 1%BSA 10%NGS (Normal Goat Serum) in PBS1X and incubate
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RT
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30min
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One by one, dry a bit each slide, surround the sections with a DAKO pen and put the slides in a humid chamber. DO NOT let the the sections to dry. Consider a V of 200ul for each slide.
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Remove the blocking solutions and incubate with the primary antibody at the appropriate dilution in 1%BSA 10%NGS 1XPBS
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4°C
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O.N.
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Incubate in the humid chamber
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Put the slides in the rack
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Wash slides in 0.1%Tween 20 PBS1X
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2x5min
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Incubate with the appropriate secondary antibody in 1%BSA 10%NGS 1XPBS
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RT
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60min
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Put the slIdes in the humid chamber. If the primary antibody is polyclonal (made in rabbit) use as secondary antibody Goat anti-Rabbit (dilution 1:400 DAKO) .If the primary antibody is monoclonal (made in mouse) use as secondary antibody Goat anti-Mouse (dilution 1:200 DAKO).
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Wash slides (in the rack) in 0.1%Tween20 PBS1X
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2x5min
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Incubatete with AB complex
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RT
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30min
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If Vectastain kit is used, prepare the solution 30’ before use and put at 4°C. For 2.5 ml put first one drop of A and then one drop of B (see data sheet of the Kit). If Dako kit is used, prepare the solution 30’ before use and put at RT.
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Wash slides in 0.1%Tween20 PBS1X
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2x5min
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In the rack
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Put the slides on 3MM paper, next to the microscope and put on them the freshly prepared DAB solution. When the signal is clear and/or before the background is too high stop the reaction putting the slides in water.
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max 10min
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After max time the reaction does not develop further.
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Stop the reaction in 50mM Tris or water
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5min
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Colour slides in Hematossilin
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30sec-1min
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Diluted 10X
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Put under water
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about 10min
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Rehydrate slides in 50% EtOH
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1x5min
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Rehydrate slides in 75% EtOH
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1x5min
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Rehydrate slides in 95% EtOH
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1x5min
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Rehydrate slides in 100% EtOH
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1x5min
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Xilene
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1x5min
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Put the slides horizontally and let them dry
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Mount the slides with DEPEX and let them dry
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dry O.N.
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DEPEX is toxic
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Clean the slides and put in the apposite box
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RT
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Other informations
DAB solution (freshly prepared and stored in dark)
Put one tablet of DAB in 5ml of 50mM Tris or bidistilled H2O. Filter.
Add 5ul of 30% H2O2 (1000X) and distribute on the sections.
Note:
In some cases, after dewaxing sections, it is advisable a heat treatment (microwave oven) in citrate buffer 0.01 M ph 6.0 to retrieve the antigen sites.
Quality validation:
Yes
Validation info
The protocol has been used to produce data published in peer-reviewed journals.
Solutions
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Block endogenous peroxidase (200ml)
Reagents F-concentration Quantity I-concentration H2O20.3%20ml30% (100x)MetOHabs180ml -
Blocking solutions (200ul)
Reagents F-concentration Quantity I-concentration BSA1%20ul10%NGS (Normal Goat Serum)10%20ul100%1XPBS160ul
