Preface:

Procedure:

•  First stain
•  HRP Developing solution
•  Second stain
•  AP Developing solution

First stain:
Indirect immunohistochemistry with avidin-biotin  peroxidase(e.g rabbit anti antigen X, goat anti-rabbit biotin, avidin HRP)

1- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added.
2- Briefly blot the slides without letting them dry and then apply 3% human or pig serum as a blocking agent (health hazard!).
3- incubate with the blocking for 10 min. If one of your antibody is biotin-conjugated, you need at this point to do endogenous biotin blocking.
4- blot the slides without washing and apply the primary antibody, in a moist chamber, at RT for 1-18 hr.
5- Wash twice in TBS 0.05M pH7.5 + 0.01% Tween 20.
6- add the biotin-conjugated secondary antibody (50 to 100 µl) and incubate for 45 min. The secondary antibody should be absorbed against human serum; if not add 1% human serum before use.  Most reagents are used at 1:100 or 1:200 dilution.
7- Wash twice in TBS 0.05M pH7.5 + 0.01% Tween 20.
7bis- block endogenous peroxidase by incubating in 0.1%NaN₃ and 0.3% H₂O₂ for 30 min. Wash thrice.
8- add the HRP-conjugated avidin (50 to 100 µl, dilution 1:300 -500 in nTBS-Tween) and incubate for 20 min. Be careful not to dilute the avidin in biotin-containing medium.
9- Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20.]
10- add 50 ml of  the developing solution (see below ). Protect from direct light.
11- after 5 min, check the staining in your positive and negative controls.
12- check the staining until complete, dense staining is obtained, but background is still low.
13- when staining is complete, wash thoroughly in tap water.
14- transfer to TBS0.05M pH7.5 + 0.01% Tween 20.

HRP Developing solution:
For 50 ml developing solution add in order:

• Aminoethylcarbazole (20 mg tablets, Sigma A-6926, dissolved in 2.5 ml NN-DM formamide)
• 50 ml acetate buffer pH 5.5 (52.5 ml of 0.1M acetic acid solution + 196.5 ml of a 0.1M Na acetate solution, bring to 500 ml)
• 25 µl H₂O₂ 30%.

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Second stain:
Double indirect immunohistochemistry (e.g rabbit anti antigen X, goat anti-rabbit AP, rabbit anti-goat AP)

A second blocking step is omitted, because blocking is due to the first stain.
One can briefly boil the slides to re-retrieve antigens and quench the primary layer. (bring to a boil in EDTA buffer and let cool).

1- apply the 2nd primary antibody, in a moist chamber, at RT for 1-18 hr.
2- Wash twice in TBS 0.05M pH7.5 + 0.01% Tween 20.
3- add the AP conjugated secondary antibody (50 to 100 µl) and incubate for 45 min. The secondary antibody should be absorbed against human serum; if not add 1% human serum before use.  SBA Goat anti mouse AP or Goat anti Rabbit AP can be used 1:200 in TBS-BSA NaN₃.
4- Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20.
5- add the AP conjugated tertiary antibody (50 to 100 µl) and incubate for 15 min. The tertiary antibody should be absorbed against human serum; if not add 1% human serum before use.  SBA Goat anti mouse AP or Goat anti Rabbit AP can be used 1:200 in TBS-BSA NaN₃.
6- Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20.
7- add 50 ml of  the developing solution (see below ). Protect from direct light.
8- after 5 min, check the staining in your positive and negative controls.
9- check the staining at 10-15 min interval.
10- when staining is complete (usually < 1 hr), wash thoroughly in tap water.
11- preferably postfix in formalin for 4-5 hrs before mounting in water soluble mounting medium (glycerol gelatin).

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AP Developing solution:
For 50 ml developing solution add in order:

• 50 ml Tris-Hcl 0.1M pH 9.2 (exact!) (1:10 from a stock solution 1M)
• Levamisole 1mM (12 mg)
• 20 mg Naphtol As BI phosphate (stock solution 40 mg/ml in NN-DM formamide, anhydrous, kept at -20°C)
• 10 mg Fast Blue BB Diazonium salt (Sigma F3378)

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