Dec 04, 2025
Immunohistochemistry _ Chromogenic_FFPE
- Yue Ma1
- 1VanAndelInstitute
Protocol Citation: Yue Ma 2025. Immunohistochemistry _ Chromogenic_FFPE. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx442dl8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 25, 2025
Last Modified: December 04, 2025
Protocol Integer ID: 230756
Keywords: ASAPCRN, ffpe sample dab, good for ffpe sample dab
Abstract
The protocol is good for FFPE sample DAB staining
Guidelines
4. Wash sections with PBST or TBST (0.1% Tween20) for 10 mins, repeat for 3 times.
5. Quenching: incubate with 0.3% H2O2 (stock 30%, 4°C, SigmaH1009) in methanol for 10mins at 4°C.
6. Wash sections with PBS or TBS for 5 mins, repeat for 3 times.
7. Secondary antibody incubation: dilute biotinylated goat anti-xx IgG (rat 1:400, rabbit 1:500, mouse 1:500) in TBST, incubate for 2h at RT or 24h at 4°C.
8. Wash sections with PBST or TBST (0.1% Tween20) for 10 mins, repeat for 3 times.
9. Prepare 30 mins before use ABC (avidin-biotin complex) mix: 10 ml PBS+ 4 drops of reagent A(vortex)+ 4 drops of reagent B(vortex), let stand at RT for 30 mins.
10. ABC incubation for 1h at RT.
11. Wash sections with PBST or TBST (0.1% Tween20) for 10 mins, repeat for 3 times. Then wash with PBS for 5 mins, repeat for 2 times.
12. Prepare DAB solution: 10 ml PBS+ 4 drops of reagent 1 (buffer PH7.5, vortex) + 8 drops of reagent 2 (DAB, vortex) + 4 drops of reagent 3 (H2O2, vortex). Revelation with DAB chromogen until grey staining is available.
13. Counter staining (optional):
- 0.1% Cresyl violet/Nissl staining: 15 mins.
- Hematoxylin staining: drop slides in Harris Hematoxylin Solution (Sigma) for 5 mins, then transfer slides to tap water for 5 mins, then 0.5% hydrochloric acid in 70% ethanol (start from 30s, keep until staining look good), then transfer slides to tap water for 5 mins.
14. Dehydrate: add slides to 70%, 95%, 100% ethanol for 5 mins each.
15. Clearance: 1min in Xylene I, then in Xylene II (use fresh solution) until mounted.
16. Coverslip mounting with Entellan (Merk) medium.
Troubleshooting
Bake slides
Bake slides in a dry oven for 1h at 60°C.
Deparaffinize and hydrate tissue sections
Incubate the slides in xylene in the fume hood for 5 min.
Place in the second xylene in the fume hood for 5 min.
Remove the slides from the xylene, and immerse them in 100% ethanol for 2 min.
Place in the second 100% ethanol for 2 min.
Immerse the slides in 90%, 80%, 70% EtOH, each for 2 min, and then distilled water for 1 min, then keep the slides in PBS until ready to perform the Antigen Retrieval. Do not allow the slides to dry out.
Antigen retrieval
Prepare antigen retrieval buffer base on the antibody’s data sheet. [Tris/EDTA buffer pH 9.0; 10x citrate buffer 0.01M, pH6; 10x HIER pH 6 (Abcam)]
Transfer the slides into the container with antigen retrieval buffer.
Place the container into the steamer.
Turn on the steamer, heat the antigen retrieval buffer to 95-100°C, start counting for 30 min.
Remove the container from the steamer and allow it to cool down for 20 min (~60°C).
Staining
Draw a circle around the tissue with Hydrophobic Barrier PAP Pen (Vector), let it dry.
Blocking: 10% normal goat serum + 0.1% Triton X in PBS or TBS for 1h at RT.
Primary antibody incubation: dilute antibody in 5% normal goat serum+ 0.1% Triton X in PBS or TBS for 24-48h at 4°C.
Wash sections with PBST or TBST (0.1% Tween20) for 10 mins, repeat for 3 times.
Quenching: incubate with 0.3% H2O2 (stock 30%, 4°C, SigmaH1009) in methanol for 10mins at 4°C.
Wash sections with PBS or TBS for 5 mins, repeat for 3 times.
Secondary antibody incubation: dilute biotinylated goat anti-xx IgG (rat 1:400, rabbit 1:500, mouse 1:500) in TBST, incubate for 2h at RT or 24h at 4°C.
Wash sections with PBST or TBST (0.1% Tween20) for 10 mins, repeat for 3 times.
Prepare 30 mins before use ABC (avidin-biotin complex) mix: 10 ml PBS+ 4 drops of reagent A(vortex)+ 4 drops of reagent B(vortex), let stand at RT for 30 mins.
ABC incubation for 1h at RT.
Wash sections with PBST or TBST (0.1% Tween20) for 10 mins, repeat for 3 times. Then wash with PBS for 5 mins, repeat for 2 times.
Prepare DAB solution: 10 ml PBS+ 4 drops of reagent 1 (buffer PH7.5, vortex) + 8 drops of reagent 2 (DAB, vortex) + 4 drops of reagent 3 (H2O2, vortex). Revelation with DAB chromogen until grey staining is available.
Counter staining (optional):
0.1% Cresyl violet/Nissl staining: 15 mins.
Hematoxylin staining: drop slides in Harris Hematoxylin Solution (Sigma) for 5 mins, then transfer slides to tap water for 5 mins, then 0.5% hydrochloric acid in 70% ethanol (start from 30s, keep until staining look good), then transfer slides to tap water for 5 mins.
Dehydrate: add slides to 70%, 95%, 100% ethanol for 5 mins each.
Clearance: 1min in Xylene I, then in Xylene II (use fresh solution) until mounted.
Coverslip mounting with Entellan (Merk) medium.