General Staining Procedure for IHC on FFPE Tissue Section Using Acu-Stain HRP kits
 
1.             Bake slides in 55 – 60°C oven for 30 minutes to overnight
2.             Deparaffinize slides in two baths of xylene, 5 minutes each
3.             Rehydrate in two baths of 100% ethanol, one bath of 95% and 80% ethanol, 3 minutes each
4.             Eliminate endogenous peroxidase in 3% H2O2 in water, 10 minutes
5.             Rinse slides in 1X PBS or water
6.             Perform appropriate pretreatment (HIER or enzyme digestion)
7.             Rinse slides 3 times with 1X PBS, 2 minutes each
8.             Apply blocking solution, incubate 10 minutes (DO NOT RINSE OFF)
9.             Apply 1° antibody, incubate 30 – 60 minutes
10.          Rinse slides 3 times with PBS-T, 2 minutes each
11.          Apply 2° antibody, incubate 10 minutes
12.          Rinse slides 3 times with PBS-T, 2 minutes each
13.          Apply enzyme conjugated streptavidin, incubate 10 minutes
14.          Rinse slides 3 times with PBS-T, 2 minutes each
15.          Apply substrate-chromogen solution, incubation time varies with each reaction (e.g. 5 minutes for DAB, 10 minutes for AEC, etc.)
16.          Rinse slides under running tap water, 2 minutes
17.          Counterstain slides in Meyer’s Hematoxylin, 2 minutes
18.          Rinse off excess Hematoxylin under running tap water
19.          Dip slides in 1X PBS 10 times
20.          Rinse slides under running tap water, 30 – 60 seconds
21.          Dehydrate slides in one bath of 80% and 95% ethanol, then two baths of 100% ethanol, 10 dips each
22.          Dip slides in two baths of xylene, 10 dips each
23.          Coverslip slides
24.          Microscopy

All steps done in room temperature, unless otherwise specified.

FFPE: formalin-fixed, paraffin-embedded
HIER: Heat Induced Epitope Retrieval
PBS-T: 1X PBS with 0.01% Tween 20