Hi Adity, it is a bit difficult to answer your question not knowing your procedure. Have you optimised your primary antibody using different dilutions of it and checking which gives you good staining? A lack of staining for your target antigen may indicate at least two things: the antigen is not present in your tissue, or the concentration of your antibody is too low.

The best procedure is to optimise your antibody on a known positive control, so you have a good reference images and then move to your test slides. Another possibility is that if you have a good staining in positive controls and almost none in your test tissue - that would indicate that whatever you are looking for is down-regulated in your test samples.

I understand that you are using a primary antibody with a fluorescent tag - works best when there is an abundant antigen in tissue - a two step method with secondary Ab with fluorescent tag ,or more complex enhancement methods, are useful if you have little expression of antigen in your samples and you need to enhance it's visibility.

So, first try optimisation on positive sample - then move to your own samples. The more complex the method, the more important is to have a clear negative control.

Good luck with your project.

Hi Adity

I am totally agree with Maria's suggestion.

But what do you mean by :

I can only see DAPI and nothing else. ?

Two step immunostaining are ideal probes for one-step staining since they are monovalent and 10 times smaller than conventional secondary antibodies. Their small size leads to just a minimal increase in size of the resulting complex with the antibody. The small size of the primary:secondary complex allows easy target access and enables fast penetration even into dense tissue. In addition, the small size of a Nano-Secondary decreases the distance between target and label, which leads to higher imaging resolution.