Yes, it is possible but I don't really recommend it. The reason is that color compared to fluorescence is not very good at discriminating between two signals. Also because you can not really quantify the signal. Fluorescence would be the way to go for the kind of dual staining you are talking about. Fluorescence has much better resolution, sensitivity and separation. The only problem with fluorescence is that you need a good fluorescence microscope.

BTW, Vector sells dual color probes for IHC.

Regards

Dear Kunal,

I agree with Nurul, and suggest you use fluorescent secondary antibodies.  However, if you don't have access to a fluorescent microscope, it is possible to do two different colors.  As Nurul mentioned, Vector has different substrates for their kits and suggestions for two color staining.

What is very important, no matter which path you take, is the quality of the secondary antibodies used for double labeling.  For the easiest method, your two different primary antibodies, which recognize your two different proteins of interest, should be made in two different species (for example, rabbit anti-protein #1 and mouse anti-protein #2).  However, your secondary antibodies should be made in the same animal (goat anti-rabbit and goat anti-mouse for the above example).  For the secondary antibodies, I prefer to purchase reagents that are designed for double-labeling and have minimal cross-reactivity with other species.  There are many threads here on RG that have hints for controls and troubleshooting of such labeling procedures.

Since you are using the ABC technique, you may find that you will need to change the concentration of your primary antibody if you switch to detection with a labeled secondary.  If this is a problem, you can still use the ABC technique with a fluorescent detection system.  I use biotinylated secondaries with a fluorescent streptavidin, or the ABC technique with FITC-tyraminde (rather than DAB), to detect the less abundant protein of interest.  I then label the more abundant protein without ABC amplification by using a fluorescently labeled secondary.

Good Luck!

Jill 

Hi again, Kunal

Here is a link to one discussion here on RG on multicolor labeling.  David has some really good hints:

https://www.researchgate.net/post/Four-color_immunofluorescence_Has_anyone_had_success_in_staining_with_four_colors_on_CNS_tissue

Best,

Jill

Thanks a lot Jill for your suggestions! Could you please provide me the catalogue number for these dual color probes from Vector labs? And would you please share your protocol for this kind of dual staining.

Thanks

Hi Kunal,

I do agree with Jill and Nurul that indeed fluorescence microscopy has a very good resolution, but only when using the right equipment and when making sure that you have proper separation of your fluorescent spectra.

With regards to bright field multiple labeling, it is a bit more tricky, but with the right tools you can get very reliable data from it, together with tissue morphology. We regularly use the labels from Vector laboratories to this end. Examples are vector red (SK-5100) combined with Immpact DAB (SK-4105). To fully and reliably separate the colors along with Haematoxilin, we make use of the Nuance FX sprectral imaging system from Perkin Elmer. Please find attached some papers regarding the technique in which they describe up to four lables simultaneously.

Good luck!

Bram

Hi there,

I understand that dual IHC staining is used quite frequently in clinical pathology, and there is no need for fluorescence microscopy. The approach there is to use antibodies originating from different species (mouse/rabbit). Afterwards the DAB is used for one of the primary antibodies, and i.e. AP for another one. The protocols could be tricky, but if everythings is working, the pictures are nice.

Peter

If you are delaing with FFPE tissues I would definitelygo for a dual blue and red chromogen-based enzyme-based permanent staining (based on AP or peroxidase), rather thanfluorescence. In case the antigens are located in the same cells) using FFPE-tissue I would try to deconvolute the chromogenic signal or as a last resort use directly fluorochrome conjugated antibodies (high risk of autofluorescence). Alternatively I would do a  artificial double staining on two adjacent sections and then stitch the images together using digital pathology (VisioPharm or HALO).

Hi Kunal,

If you read the van der Loos 2010 J Histochem Cytochem paper that Bram attached, you will learn everything you need to know to make an informed choice for your detection systems.  (Thanks, Bram for that.  I only had the 1988 and 1989 van der Loos papers for the protocols I used to use!).  If you choose to use the Vector products, there is a lot of information on their website.

https://vectorlabs.com/protocols/

Unfortunately, there is not a one-size-fits all protocol.  Your protocol will depend on your primary antibodies, and the system of secondary antibodies that you choose to use, as well as the way that the tissues were processed for sectioning.  I use frozen sections and fluorescent labels because that is the easiest for me to use to get good results, and I have access to both a confocal and multiphoton microscope setup.

Good Luck!

Jill

Thank you so much guys! I got the necessary information.