DAB HRP Substrate-Chromogen ( 2 Liquid Components Kit )
This kit is supplied in 2 liquid components
3A-(Buffer+ substrate) 10X conc. --- 10 mls
3B-DAB 50X Chromogen --2ml
To 0.2 ml of 10X conc. 3A (Buffer+substrate) Reagent, add 1.8 mls of distilled or deionized water in mixing bottle or test tube, mix well, add one drop (40 microliters) of 3B Chromogen Reagent, mix well.
This ready-to-use reagent is good for 2-4 hours at Rt or 6-8 hours at 4C.
STAINING PROCEDURE FOR IHC
1. Wash tissue slide thoroughly with buffer (no azide in this buffer ).
2. Apply this solution to the specimen.
3. Incubate for 15-20 mins at RT or 37C.
4. Wash specimen with distilled or deionized water 5-7 times.
5. Counterstain. We normally use hematoxylin (Biomeda M10) 30-60 seconds. Wash thoroughly with tap water, buffer pH over 7.4, followed by washing with distilled or deionized water.
6. DAB can be mounted with our aqueous mounting medium Crystal mount ( Biomeda cat. # M02) or after dehydration with gradients of alcohol and toluene or xylene or any other dehydrating agent, followed by mounting medium (Biomeda Clarion Cat. # M05 ) or Permount.
STAINING FOR WESTERN BLOT
This mixture can also be used in Western blot. It may be necessary to dilute ready to use reagent 1:2 or 1:5 with diluted buffer. Incubate, as soon as brown spot appear, stop the reaction by washing with buffer, followed by distilled or deionized water.
THE OPTIMUM TIME OF INCUBATION AND DILUTION SHOULD BE DETERMINED BY THE INDIVIDUAL LAB.
Liquid 2 components
2-8 C. Do NOT FREEZE