Double Immunohistochemistry

 

Preface:

You should not co-stain in IHC antigens located on the same structure (nucleus, membrane, cytoplasm). That because the success of this double staining procedure is based on the complete development of the first stain, which should mask the structure stained first. Therefore you should be able to use two antibodies raised in the same species. However better results are obtained if two different species are used.
Careful check possible crossreactivty of your secondary antibodies by staining in simple IHC the relevant heterologous primary antibodies. E.g. many goat anti mouse IgG ₁ heavy chain do stain rat IgG _2a and IgG _2b .

 

 

Procedure:

Apply the correct procedure (fixation or antigen retrieval) to the sections.
Apply the blocking procedure. Be aware not to use for blocking any component which may be detected by any secondary antibody you plan to use.

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%NaN ₃ and 0.3% H ₂ O ₂ 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 H ₂ O ₂ 30%.

Shake well
Filter with a 45µm filter (optional).
Keep away from direct light, use within 5 min.

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 NaN ₃ .
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 NaN ₃ .
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).

Do not counterstain, unless you can afford a very gentle hematoxilyn hue in the nuclei.

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)

Shake well
Filter with a 45µm filter.
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