Method: IHC staining of paraffinized tissue for histamine receptors
1. Deparaffinization & hydration
Xylene for 5min x 3 times (I, II, III)
100% ethanol for 1min x 3 times (I, II, III)
95% ethanol for 1min;
70% ethanol for 1min;
DI water for 5min x 3.
2. Antigen Retrieval with Citrate Buffer DAKO Target Retrieval solution
Prepare Antigen Retrieval (AR) solution by diluting a stock 1:10 in DI water.
Heat up waterbath to 99C and put AR solution into the heated waterbath.
Bring this AR solution to 80-85C before placing slides into solution.
Place into heated working citrate buffer for 30min.
Allow to cool, in citrate buffer, for 30min.
Rinse well with DI water
3. Staining
Peroxidase Block (H2O2) for 25min.
Rinse in TBST – 5min x 3
Cycle the section with PAP-pen
Avidin Application –10min
Rinse in TBST – 5min x 3
Biotin Application –10min
Rinse in TBST – 5min x 3
Protein Block – 30 min
Primary antibodies (HRH1 Ab – 1:100) – overnight at 4C.
Rinse in TBST - 5min x 3
Biotinylated Secondary Anti-Rabbit Antibody 1:200 (from Vector Laboratories, CA) – 30min at RT
Rinse in TBST – 5min x 3
Streptavidin-HRP – 10min
Rinse in TBST – 5min x 3
4. DAB application
Add 1 drop (or 20µL) of the DAB chromogen per ml of Substrate Buffer, mix well (stable within 5 days at 2-8⁰C). Apply using transfer pipette (5 min).
Rinse gently with DI water.
5. Counterstaining with hematoxylin
Hematoxylin – 30-60 sec (or more).
Rinse with DI water
0.08% ammonia hydroxide in water (bluing reagent) until stain is blue ~30sec
Rinse with DI water
Reagent alcohol, 95% - 1-2min x 2
Absolute ethanol – 1-2min x 2
Xylene - 1-2min x 2
6. Mounting with a mounting media and coverslip