What to prepare: KIT VOLUME INCUBATION WASHING

Peroxidase 3 drops 5 minutes Wash buffer

A. Her-2 antibody – 5uL Ab concentrate + 1495uL IHC diluent block (yellow)
(1:300) Protein block 3 drops 5 minutes none
B. Bond wash – 100mL Bond wash concentrate + 900mL (green)
distilled water Primary Ab 150uL 30 minutes Wash buffer
C. ER1( PH6 ) / ER2 ( PH9 ) – 5mL ER solution 45mL distilled Post primary 3 drops 30 minutes Wash buffer
(red)
water
Compact 3 drops 30 minutes Wash buffer
D. DAB – 10uL DAB chromogen + 200uL DAB substrate buffer;
polymer
incubate for at least 5 mins before applying. (purple)
E. Decloaker – fill with 500mL of water DAB (brown) 2-3 drops 10 minutes Deionized
F. Humidity chamber – to be used during incubation phase water
Hematoxylin 3 drops 5 minutes Deionized
(blue) water

IHC MANUAL PROCEDURE: LEICA NOVOLINK

6. Dehydration – 95% alcohol 5 mins / 100% alcohol 5 mins
1. Baking – 60C for 30mins 7. Clearing – 2-3x changes of xylene for 2 mins
2. Dewaxing – 2-3x changes of xylene for 2 mins each 8. Mounting
3. Rehydration – 100% alcohol 5 mins / 95% alcohol 5 mins
4. Epitope retrieval
a. Enzyme pre-treatment kit DESMIN
b. No retrieval PANCYTO
c. Heat induced epitope retrieval – ER1 (pH 6) and ER2 CD5
(pH 9) CD20
i. Separate slides according to retrieval pH CD45
ii. Heat retrieval using decloaker for 40 mins
@ 95C ( cool down for 10 mins )

5. Detection kit