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ab41621: Abreview - how was direct fluorescence done?
ab61859: IHC-P protocol
Asked on Sep 12 2012
Thank you for contacting us.
I have contacted the Abreview customer regarding more information about the staining with ab41621. I will let you know if I hear back from her.
As for ab61859, I have the following IHC-P protocol:
General IHC Protocol
1. Deparaffinize sections in xylene, 2x5min.
2. Hydrate with 100% ethanol, 2x3min.
3. Hydrate with 95% ethanol, 1min.
4. Rinse in distilled water.
1. Follow procedure for pretreatment as required.
2. Procedure for Immunoenzyme Staining.
3. Rinse Sections in Washing Buffer (PBS+0.1% Triton X-100, pH 7.4) for 2x2 min.
4. Serum Blocking: incubate sections in normal serum block – species same as secondary antibody( Wash Buffer + 5% Normal Sera). Note: since this protocol uses avidin-biotin detection system, avidin/biotin block may be needed based on tissue type. If you do, the avidin/biotin blocking should be done after normal serum block and before primary antibody incubation.
5. Primary Antibody: incubate sections in primary antibody at appropriate dilution in Wash Buffer for 1 hour at room temperature or overnight at 4C. Note:Do not rinse sections between serum block and primary antibody incubation.
6. Rinse in washing buffer for 3x2 min.
7. Peroxidase Blocking: incubate sections in peroxidase blocking solution for 10 minutes at room temperature.
8. Rinse in washing buffer for 3x2 min.
9. Secondary Antibody: incubate sections in biotinylated secondary antibody at an appropriate dilution in Wash Buffer for 30 minutes at room temperature.
10. Rinse in washing buffer for 3x2 min.
11. Detection: incubate sections in streptavidin-HRP in Wash Buffer for 30 minutes at room temperature.
12. Rinse in washing buffer for 3x2 min.
13. Chromagen/Substrate: incubate sections in peroxidase substrate solution.
14. Rinse in washing buffer for 3x2 min.
15. Rinse in running tap water for 2-5 minutes.
16. Dehydrate through 95% ethanol for 1 minute, 100% ethanol for 2x3min.
17. Clear in xylene for 2x5min.
18. Coverslip with mounting medium.
0.3% H2O2 in PBS (for Paraffin Sections)
0.3% H2O2 in Methanol (for Frozen Sections)
0.001% Avidin in PBS
0.001% Biotin in PBS
Block for 10 minutes with first solution, rinse with PBS, block for 10 minutes with second solution.
I am checking with the lab regarding the antigen retrieval method used with this antibody.
I hope this information is so far helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Answered on Sep 12 2012