General Staining Procedure for IHC

General staining procedure for Genemed Immunohistochemistry Products. Visit the procedure for answers to many common staining procedural questions asked by customers.

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

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