This is the classical reason for using negative controls in IHC. Please look for literature, that describes the use of controls in IHC. The main thing is, that you have to omit the primary antibody, so any seen fluorescence in the negative control is an artefact (or autofluorescence).

I agree with Gudrun Lang.

I do not completely understand why you say that the fluorescence you see is non specific.

If you see it with your cells/tissue without having performed any IHC (only mounting) then it is autofluorescence.

If you see it after doing IHC omitting your primary antibody (if there is a system with primary antibody followed by secondary antibody conjugated to FITC) then it is unspecific binding of your secondary antibody which can be eliminated by blocking your slices better.

If your antibody is not commercial and was obtained in your laboratory and is polyclonal maybe you can analyze it specificity on a Western Blot (it should be very specific to use for IHC).

If you have available cells/tissue that you know not to express NMDAR or/and GR you can use them to probe your antibody and should obtain a black image.

Hi Nagar,

I agree with the above comments; In addition, I would like to point out that if you're fixing any cells/tissues in you're experiments, then there's a propensity to generate artifacts (esp if you use harsh fixation steps). Therefore, it is advisable to optimize your protocol for the fixation step and of course use negative controll experiments.

This is again one of these questions that is virtually un-answerable. This is not meant in a negative way but I would like to point out that many questions here on Researchgate are very vague. 

To determine whether an immunofluorescence staining is background, autofluorescence or real, one has to perform a few controls. This was already pointed out correctly by the others.

Autofluorescence: just include a slide/section to which you don't add antibodies and secondaries. 

Omit primary to test the background of the secondary system. This should be really minimal if you use commercial stuff. The secondary is generally really not the problem unless there is a mix-up with the species, like you use anti-mouse secondaries on mouse tissues...

The problem is mostly with the primary antibody. There i little you can do to figure out whether your primary is good or bad. You can use a normal serum from the same species your primary is made in but that tells you very little. Nice control but it only shows the really general background, which should be minimal.

To figure out whether your antibody is good, you would need another antibody against the same protein, best different epitope, and get the same result. This is in my opinion the brutal truth about immunostainings. I have no idea how many epitopes are on a tissue section and how they relate to the epitopes on a Western, a frozen section, cell culture cells, formalin-fixed cells, antigene retrieval treated sections... I assume there are rally a lot of epitopes on a section, some denatured, some unaccessible, some modified, some similar to the one in the protein of interest but in an other unrelated protein.

One can make a lot of assumption about this but in the end the specificity of the primary antibody is difficult to definitely prove. Most of the time, we trust the source, hope they tested well, have evidence that the staining pattern is in accordance with all available data AND another antibody against the same protein shows a damn similar staining pattern.

Simple answer: do the appropriate controls, evaluate the staining and whether it makes sense. Controls are half of your experiment, 45% is labeling and the rest is the actual experiment (that is what I tell everyone in the lab).

Complicated answer: even if all your controls are fine, even if you have the peptide that was used to raise the antibody and use it as a negative control blocking reagent, even if the antibody is perfect in Western blotting, it is very still hard to be be 100% sure that the signal is real. But being close to 100% is often fine and with some circumstantial evidence (proteins is nuclear, cytoplasmic etc. is expressed highly in this cell type and not another etc.) one get to make a pretty confident statement about the staining.

Before you do any IHC work using anti-GR antibodies, you have to be aware that not all antibodies are equal, and often they will give what appears to be unspecific binding.

Remember that the GR is about the only ubiquitously expressed receptor. It has been found in all tissues and cell types investigated. It has membrane forms (but they represent a tiny fraction), and is mostly cytosolic. In theory you should be able to see it translocating from the cytosol to the nucleus, however, this is rarely the case. Often, if you don't fix cells properly, you get what appears to be unspecific binding.

Have a good read of Angela Sarabjitsingh's excellent paper:

Brain Res. 2010 May 17;1331:1-11. doi: 10.1016/j.brainres.2010.03.052.

Specificity of glucocorticoid receptor primary antibodies for analysis of receptor localization patterns in cultured cells and rat hippocampus.

Sarabdjitsingh RA1, Meijer OC, de Kloet ER.

Hope this helps

Jon

Thanks to all