Anti-CD68 Immunohistochemistry staining protocol
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 99⁰C and put AR solution into the heated waterbath.
Bring this AR solution to 81-85⁰C before placing slides into solution.
Place into heated working citrate buffer for 27min.
Allow to cool, in citrate buffer, for 30min.
Rinse well with DI water
3. Staining
Peroxidase Block (3% H2O2 ready solution) for 25min.
Rinse in TBST – 5min x 3
Cycle the section with PAP-pen
Avidin Application –11min
Rinse in TBST – 10min x 3
Biotin Application – 11min
Rinse in TBST – 10min x 3
Protein Block – 30-60 min at RT or overnight at+4C
Primary mouse α-CD68 Antibody1:50 (sc20060) – overnight at 4C or 2 hr at RT.
Rinse in TBST - 10min x 3
Biotinylated Secondary Anti-MouseAntibody (BP-9200, Vector Labs) – 30-60 min at RT
Rinse in TBST – 10min x 3
Streptavidin-HRP – 30min !!!
Rinse in TBST – 10min 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 (25-30min).
Rinse gently with DI water.
5. Counterstaining with hematoxylin
Hematoxylin – 30-60 sec
Rinse with DI water
0.08% ammonia hydroxide in water (bluing reagent) until stain is blue ~30sec
Rinse with DI water
6. Dehydration
Reagent alcohol, 95% - 1-2min x 2
Absolute ethanol – 1-2min x 2
Xylene - 1-2min x 2
7. Mounting with a mounting media (Vectashiled without DAPI) and coverglass