Immunohistochemistrv StaIning

By Woody CU HK

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This is basicly the ABC method of IHC.

1.- Incubate in Autoblot oven for 30 mm. @ 56 oC.

2.- Check that solutions are full.
3.- Bring Xylene to oven -- Do not let tissues solidify.

4.- Dip in solutions for 5 min. each to hydrate:
a.Xylene
b.Xylene

c.100%alcohol
d. 95% alcohol
e.85% alcohol (No Stock-might need to make)

f. 75% alcohol (No Stock)
g. 50% alcohol (No Stock)
h.Distilled water
5. - Bring slides in water to bench.

6.- Wet incubater.
7.- Dry slides carefully -- Do not touch tissue.
8.- Add .1% Hydrgen Peroxide~H2O2) and with plastic dropper and

incubate for 10 min
9.-Dry slides on paper towels. Put in PBS for 5 mm..

10.-Dry slides again..
11.- Put slides in special slide holder filled with .01 M Citric Acid Buffer.

12.- Place slide holder in partly full water beaker.
13. -Microwave for 5 mm..
14.- Add more buffer if necessary. Microwave for another 2 mm..
15.- Mix 1 drop of Horse Serum with 1 ml of PBS in kit mixing bottle on ice
16.- Let slides- cool. Put in PBS for 5 min.
17.- Dry off slides. Incubate with enough Horse Serum Solution to

cover tissue for 20 min.
18.- Wash in PBS with stir bar for 5 min.
19.- Change PBS quickly, rewash for another 5 min..
20.- Mix 100 micro-liters Primary Antibody with 400 ul of 1 % PBS-BSA.
21.- Add~50 micro-liters per slide.
22.- Let incubate for 2 hours.
23.- Change PBS, wash slides for 5 mm..
24.- Rewash 2 more times with new PBS each time.
25. -Mix 1 drop stock Secondary Antibody (Biotinylated) with 1 ml PBS in mixing bottle.
26.- Add Secondary antibody to slides (enough to cover samples). Incubate for 30 mm..
27 - - Prepare ABC in mixing bottle: It should sit on ice for 30 mm. Add 1 drop s olution A tol ml PBS in mixing bottle. Mix.
28 Add 1 drop of solution B to mixing bottle.
29 Wash slides three times in PBS, 5 mm. each.
30 Incubate slides for 30 mm. with a drop of ABC solution on each slide.
31 Wash 3 times for 5 mm. in new PBS (renew after each wash).
32 Immediately before use, to 5.0 ml of distilled water, add 2 drops of
DAB Buffer Stock Solution and mix well.
33 Add four drops of DAB stock solution and mix well.
34.- Add 2 drops of the Hydrogen peroxide Solution and mix well.
35.- Dry slides.
36.- Incubate slides with DAB solution for 3-7 mm..
37. - Wash for 5 mm. in distilled water.
38.- For 30 sec. each, dip in the following solutions to dehydrate:
a.50% alcohol
b.85% alcohol
c.95% alcohol
d.100% alcohol~- -
e.Xylene (not same solution as used in beginning);
f.Xylene
39.- Mount coverslip using a permanent mounting medium (Permount).