IMMUNOHISTOCHEMISTRY

                IMMUNOHISTOCHEMISTRY

0.001M Anhydrous citric acid buffer solution pH - 6.0

1.92 gm of Anhydrous citric acid is dissolved in 50 ml of distilled water. 7.5 ml of 4N Sodium hydroxide is added and pH is adjusted to 6.0.

The solution is made upto 1 liter by adding distilled water.

0.05 Tris buffered Saline pH - 7.6

6.1 gm of Tris base (Tris hydroxy methyl amino methane) and 8.76 gm of sodium chloriue is dissolved in 50 ml of distilled water.

37 ml of 1 N HCl is added to it.
It is made up to 1 liter with distilled water.

The pH is adjusted to 7.6 using either sodium hydroxide or hydrochloric acid.

DAB Chromogen
One drop of DAB chromogen is added to 1 ml of substrate buffer provided in the kit.


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Procedure

1)   Keep the slide in Xylene I -5 mts.
2)   Keep it in Xylene I -5 mts.
3)   Add ().5% hydrogen peroxide in methanol for 30 mts.
4)   Rinse with Tris buffered saline.
5)   Transfer the slides into 0.01 M anhydrous citric buffer in a coplin jar.
6)   Microwave at power level 80 for 5 mts
7)      Cool it for 2-3 mts. .
8)     Microwave at power level 80 for 3 mts.
9)   Cool the slides for 15 mnts.
10)Treat with distilled water for 2 mts. 2 changes
11)Treat with Tris buffered Saline for 2 mts, 2 changes
12)Treat with 3% skimmed milk in Tris buffer for 30 mts. .
13)Add Primary antibody and keep it for 1-1 % hrs.
14)Treat with Tris buffered Saline for 2 mts. 2 changes.
15)Treat it with skinned milk powder for 3 mts.
16) Add secondary antibody (link antibody) and keep it for 30 mts.
17)Treat it with Tris buffered saline for 2 mts. changes.
18)Treat with skimmed milk for 3 mts.
19) Add tertiary antibody (streptavidin) and keep it for 30 mts.
20) Treat it with Tris buffered saline for 2 mts. 2 changes.
21)Add DAB (chromcgen) and keep it for 5 mts.
22) Treat with distilled water for 2 mts.
23) Counterstain with Iaematoxylin for 1-2 mts.
24)Wash it in running tap water, dehydrate, clear and mount






Preparation of silane coated slides

1.     Keep the plain glass slides in acetone for 2 mus.
2.    Transfer the slides to 1% silane in acetone.
3.    Dip the slides in distilled water
4.    Allow the slides the dry, use slide holder, so that both sides of the slides can he coated.
5.    Keep the slides with slide holder at 37°C in an incubator overnight.
6.    Transfer the slides with the slide holder to the incubator at 56°C for 24 hrs.
7.     Allow the slides to cool, cover the slides with aluminium foil and store it inside he fridge.


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