Since both primaries are rabbit, your secondary will bind to both indiscriminately, and both will be stained with DAB, as you see in your picture. Therefore it is not possible to discriminate between both markers. You say that one marker is nuclear-only and the other cytoplasmic-only, but this is what IHC is usually used for, to demonstrate and prove the subcellular localization of staining. In your case you are assuming that the brown staining in the nucleus is due to the nuclear marker, and the brown staining in cytoplasm is due to the cytoplasmic marker, but there is no way to actually prove it with such a technique. I suppose it depends what your objectives are for this staining, but ultimately I don't think any reasonably knowledgeable reviewer would accept your technique in a publication.

Usually when staining for two markers, you would do double-labelling; with primaries from different species, (and different secondary for each primary, which shouldn't cross-react with the other species), and use two different chromogens, usually DAB and a red or purple chromogen. 

Better do them separately if you cannot afford other systems. As Jean-Martin Lapointe said: for double stain, you will have to use

a) primaries derived from different species

b) secondaries conjugated with different enzymes (i.e. peroxidase and phosphatase)

Thus, you can achieve double labeling where you can also evaluate the levels of protein load (expression) in the cells of interest.

The specificity, sensitivity, retrieval conditions, concentrations etc., are matter of other discussion.

Hi Mohamed,

In general achieving good results in double staining when primaries were made in the same host is quite challenging. I never tried it in tissue but I did it in in vitro stainings with acceptable results.

The following is a protocol adapted from recommendations from Jackson immunoresearch http://www.jireurope.com/technical/fab-blok.asp

Caution, this was performed using regular Alexas conjugated secondary antibodies in Immunofluorescence assays.

>Check your primaries in independent experiments in order to determine which is the more specific/stronger from both of them. Here strong also means good antibodies directed to prominent proteins like actin, tubulin, neurofilaments, etc.

>After permeabilization/blocking go with the less strong antibody followed afterwards for the chosen secondary antibody.

>After washing the secondary do a mild fixation of  your samples (10-15 min of PFA 4% room temperature-time depends on sample thickness). This will help to hide some reactive epitopes from the first 1ry-2ry pair. Then wash, quench PFA with NH4Cl 50nm or glycine 0.1M for 30-60 min. Wash again.

>Block again 1h and proceed with the second 1ry-2ry pair of antibodies.

Hope it helps!

Best

Hi Mohamed,,

I think it is better to look for primary antibodiesd from different species

I agree with Manal 100%. Get the 2 primary antibodies from 2 different species and instead of following DAB technique, follow immunofluorescence which will clearly give you images/signals of 2 different colors/localizations giving answers for the questions you are looking for. Good luck.

You will have to use two different chromogens ( i.e.: DAB and AEC or or DAB and Nuclear Fast Red) and two different developpers ( ICC and immunoalkaline phopsphatase). Double immunohistochemical stain!

Normally, for Western you need primary antibody at a lower dilution than required for IHC.  So, a titration with different antibody concentration is preferred.  It is easy to do.  Load the control sample in all lanes.  Cut the blot into many pieces so that each will have two lanes.  Blot them with different concentrations of primary antibody.

I  think it is not wise to use two primary antibodies.

I think that the design of your experiment should be modified as you use two primary antibodies raised in rabbit. In any case using a secondary antibody will label the two together, even if the targets are very different with reference to localization. 

May be try to incubate with a first one and then use secondary antibody , DAB to reveal (but you have to rinse extensively after the last incubation with the secondary antibody to eliminate any excess of non conjugated antibodies)

In a second step use the same procedure with the second primary antibody,. You can keep your DAB revelation but you have to intensify your mixture by adding cooper or other metal to have two different staining.

I'm assuming your 2 Rbs primary antibodies will be used on paraffin sections. Yes you can use two rabbits or two mouse primary antibodies.  I've done the very same thing using a same day IHC protocol on paraffin + (polymer). However, I've if you want to use the ABC method it will take 3 days to finish instead of one. Test the following protocol (remember to insert the necessary PBS/0.05% Tween 20 washing steps for the 1st primary antibody (I've not included in the following protocol).

After antigen retrieval, quenching & milk block (for non-specific staining). Incubate in 1st primary antibody (titer?) either 4C or RT - overnight. Next day, add your biotinylated horse anti-mouse or rabbit (depends on host of 1st primary) titer 1:300 or 1:400 - RT - 1 hour.

After, incubate in Vectastain (ABC (HRP) - 1 hour. After washing, develop in Vector DAB SK-4100) or any other HRP chromogen. If using DAB stop development using tap H20 & rinse in DH20 -briefly. Incubate sections in Biocare Medical Denaturing Solution DNS001H (1:3) - 3 to 5  minutes for paraffin. This step will ensure that the second staining will not cross react with the 1st primary staining protocol.

After, need to wash using TBS/0.05% Tween 20, & incubate in 2nd primary mouse or rabbit antibody (titer?) either 4C or RT - overnight. Next day, incubate in the 2nd bintinylated horse anti mouse or rabbit either 1:300 or 1:400 - RT - 1 hour. Then incubate in working ABC (AP) Vector AK-5000 @ rt - 1 hour. Wash in TBST 3x - 2 mins each. Then use any AP chromogen e.g. Vector Red lovely deep red (chromogen only stable for 30 minutes, so antibody titers have to be right on & use a microscope to monitor chromgen staining. Wash using TBS, rinse w/tap H20 & optional counterstain using hematoxylin. Now after hematoxylin, blue using TBS & wash in tap H20 3x & running tap H20 - 20 seconds. Then while slides are in staining rack, drain & gently shake H20 off slides, then place in oven 60C-65C - for 20-25 minutes to dry. Cool slides & cover slip.

Good luck.

Yes we can make double staining with 2 primary antibodies simultaneously to co-localize a target protein with the cell of origin

An alternative approach is to use fluorescent conjugates of the two primary antibodies. 

Article Immunohistochemistry and Multiple Labeling with Antibodies f...