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%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%.

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 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).

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. Keep away from direct light, use within 5 min.

In theory the primary antibody will be specific to its target. The problem comes with the secondary antibody. When using an anti rabbit (in this case), it will not distinguish between your two primary antibodies binding to them both in a non-discriminatory way. So you won't be able to distinguish between your TOM20 and LC3 because they will appear with the same signal.

Hi Jonathas:

It is a bit tricky if both Abs from the same species, which may confuse the 2nd Abs, there are a few ways to get around it:

1) To choose fluorescence conjugated primary Ab, which avoid 2nd Ab staining, but compromise signall intensity;

2) To use two rabbit Abs consist of different light chains, and then apply 2nd fluorescenced Ab recognize light and heavy chain respectively;

3) To choose two primary Abs with different subclass, and use 2nd Ab recognize them differently.

In general you could try to separate the staining into two steps (First step: 1. primary ab + secondary ab, second step: 2. primary ab + secondary ab). If you use differently labelled secondaries and matching filters in your microscope, it might work. If you know where you expect a reaction, this might be okay for a qualitative result, although probably not suitable for publication or quantitative interpretation.

Keep in mind: If your first primary is not completely saturated with secondary antibodies in the first step, the secondary from the second step will bind there as well, leading to false positive impressions. And you might run into a steric hinderance for the second step if the antigens are too close together, giving you too low signals for that step.

Another crude idea, although I don't know if there are protocols for that and if that works: If you combine primary 1 + secondary 1 (e.g. red label) and primary 2 + secondary 2 (e.g. green label) in separate tubes before adding those complexes to your section, the complexes might bind to the section via the Fab parts of the primaries and are already differently labelled to be discriminated with the respective filters.

Nevertheless the best way would be to either go for different primary antibody hosts, label at least one of the primary antibodies directly (there are kits available for that) and use that in the second step or to use two seperate sections.

Yes you can stain two different antigens using respective antibodies produced in the same species. After blocking, incubate first primary antibody of your choice subsequently with its secondary antibody and wash and then incubate the second primary antibody of your choice and then with a different secondary antibody with different flurochrome, wash, mount and observe. You need to use different flurochromes according to the filters in your microscope and incubation must be done only after completing one antibody.

Since both are in rabbit (and most likely both polyclonal), your best option is to make dye conjugates of both primaries. Thermo Fisher has kits that make it easy to do this. Then you would label the sample with both conjugates at the same time without risk of cross-labeling (like you would with secondaries, even if you did them sequentially). The drawback is that it takes a bit of extra time to make the conjugates (though you can store what you don't use) and the label is not as bright as using secondary labeling.

Please do not try to do this. You will only loose valuable time and money for the trial and error testing phase only to find out that you have horrible background and mix up problems.

The solution is the following:

1. Do separate single channel staining for the antibodies in samples that are similar and then compare their distribution, intensity etc.

2. Try to find at least one alternative monoclonal antibody of course mouse. Remember monoclonals will always give you a very clean background.

3. You can label the primary polyclonal antibodies with different fluorophores with kits available from different companies. For example label one antibody with Cy3 and the other with Cy2. That way, you will not need any secondary antibodies. But the labeling procedure is difficult and you have to be an expert to do it and the signal in primay labeled antibody is quite low.

My suggestion is go for No. 1 or 2 of the above.

Regards

Perhaps this is simplistic, but trying each primary antibody by itself with the rabbit condary would help to determine optimal concentrations Then you could see whether doing them simultaneously or sequentially interferes with the localization and intensity.

Good luck

Double immunoenzymatic labelling procedures for the localization of antigens on cells in tissue sections using horse dish periodase (HRP) and alkaline phosphate have been descibed previously,but mainly detecting antigens and on different cells.with this type of staining when two antigens are present on the same cells,an optimal colour of each enzyme substrate product is difficult to achieve and the interpretation of their mixed color intermediate is subjective .we present a method for simultsneous demonstration of 2 antigens on the same cell.the method can be used label either single cells in suspension or cell in paraffin fixed ,tissue using combination of a particular label ,collidal immunogold silver,and about enzymatic label HRP-DAB .

The method is easily to perform and utilises commercually available staining kit.

Keywords

Dual immunohistochemical labelling ,lmmunogold silver,Horseradish perocidase,.

Msterials for the staining

HRP,horseradish peroxidase ,PBS ,phosphate buffered saline ,PBMC,peripheral blood monoclonal cells,NSS,Normal saline serum,DAB,diaminobenzidine tetrahydrochloride ,AEC-3 amino-9-ethylcarbazole ,FITC flourescein isothiocyanate,PE phycoerythrin RITC

It is, indeed, a challenging project.

I agree with Kabir. Most of the other suggestions are inappropriate and impractical.

There are several ways to do this. The standard is to use a tyramide conjugated to a fluorochrome. Such systems are commercially available from Akoya and other companies and several companies sell a wide range of such tyramides, e.g., Biotium.

The trick is to use a HRP-conjugated secondary (preferentially a polymer, e.g. from Vector Labs' ImPress Polymer reagents).The HRP will use the tyramide to generate a radical that "instantaneously" reacts with the tissue and localizes the fluorochrome covalently to the tissue. Since the fluorochrome is now attached to the tissue, the antibodies and secondaries can be washed or cooked off the slide and another primary antibody can be applied (see, e.g., protocol from Cell Signaling: https://www.cellsignal.com/contents/resources-protocols/fluorescent-multiplex-immunohistochemistry-(mihc)-with-tyramide-signal-amplification-protocol/fluorescent-multiplex-ihc-w-tyramide OR from protocol from Bethyl: https://www.bethyl.com/content/protocol-multiplexing).

This technique can be relatively easily implemented by any lab with basic knowledge of immunofluorescence and immunohistochemistry stainings.

A lot depends on the sequence of the antibodies and how the epitopes behave upon multiple "cooking" cycles. In the case here, just one additional cooking is needed which can be tolerated by many antigens/epitopes.

Label with the first antibody, then secondary HRP conjugate to that one, do a TSA reaction with the color you want. Then remove the primary antibody with heat in citrate buffer or a strong detergent BME in 10%SDS @ 56 C for 30 mins (see my paper for complete details on how to cook Article A manual multiplex immunofluorescence method for investigati...