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Immunohistochemistry (IHC) for In-situ Hybridization (ISH)-stained Mouse Sections (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Chad Vezina

Keywords

[‘in situ hybridization’, ‘probe generation’]

Subjects

[‘Gene expression analysis’, ‘Immunological techniques’, ‘Molecular biology’]

Release Date

2017-08-07

Abstract

Intended for 50-80 micron free-floating mouse tissue sections that were ISH-stained with BM Purple and post-fixed in 4%. All washing steps should be performed with gentle agitation on an orbital shaker.

Procedure

Day 1

1. Remove the 4% PFA and wash sections for 2 X 5 min at 25°C in TBS containing 0.1% TritonX-100 (TBSTx).
2. Incubate sections for 5 min at 25°C in 3.0% H2O2 in 1X TBSTx.
3. Incubate sections for 25 min at 25°C in 0.3% H2O2 in 1X TBSTx.
4. Wash sections 4 X 5 min at 25°C in TBSTx.
5. Incubate sections for 20 min at 25°C in avidin blocking solution (Vector Labs# SP-2001).
6. Rinse sections briefly with TBSTx.
7. Incubate sections for 20 min at 25°C in biotin blocking solution (Vector Labs# SP-2001).
8. Wash sections 2 X 4 min at 25°C in TBSTx.
9. For IHC with primary antibodies made in mouse species (for non-mouse primary antibodies, skip to step #10):
- A. Incubate sections for 1 hr at 25°C in a working solution of M.O.M. Mouse IgG Blocking Reagent (Vector Labs #MKB-2213).
- B. Wash sections for 2 X 3 min at 25°C in TBSTx.
- C. Proceed to step #11.
10. For IHC with primary antibodies made in non-mouse species:
Incubate sections for 1 hr at 25°C in TBSTx containing 1% bovine serum albumin (Fisher #BP1600-100), 5% heat-inactivated serum from host-animal species of secondary antibody, and 1% Blocking Reagent (Roche Applied Science # 11096176001, diluted from a 10% stock solution of Blocking Reagent dissolved in maleic acid buffer as directed by the manufacturer). This Blocking Buffer is referred to as RBTx buffer.
11. Incubate sections overnight at 4°C in RBTx buffer containing primary antibody (concentration determined empirically).

DAY 2

12. Remove primary antibody and wash sections for 6 x 5 min at 25°C in TBSTX.
13. Incubate sections for 1 hr at 25°C in RBTx containing biotinylated goat anti-mouse, goat anti-rabbit, or rabbit anti-goat secondary antibodies (Vector Lab # BA-9200, BA-1000, BA-5000, concentration determined empirically).
14. Towards the end of step #13, dilute avidin biotin complex (ABC) reagent (Vector Labs # PK6100) in TBSTx according to the manufacturer’s instructions and incubate for at least 30 min at 25°C.
15. Wash sections for 6 x 5 min at 25°C in TBSTx.
16. Incubate sections 45 min at 25°C in diluted ABC reagent.
17. Wash 6 x 5 min at 25°C in TBSTx.
18. Prepare 3, 3’-diaminobenzidine (DAB) solution (Vector Labs # SK-4100) in TBSTx as described by the manufacturer.
19. Remove sections from tube and place in petridish. Remove TBSTx and add 1X DAB solution onto sections and monitor under microscope for color development (appr. 1-10 min).
20. As soon as color becomes visible, dilute the DAB with excess TBSTw and post-fix in 4% PFA.

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium