Hi Felix,

Where should be the antigen? Me, Cy, Nu, NuMe? Did you try it w/o any retrieval, since your section is more or less native?

Have you already tried to centrifuge the antibody solution before applying on the section. It might help.

Centrifuging the antibodies (specially the secondary) can really help getting rid of these clumps. On top of that I would make double sure that you are using both primary and secondary antibodies in the optimum concentration. It seems that you have some background, which suggests you could use them more diluted.

By the way, which blocking buffer are you using? Are you diluting your antibodies in it?

Thank you all for the answers. 

Ivan, we are expecting to see perinuclear and nuclear labeling (since it's a transcription factor). We tried this experiment with and without retrieval, unfortunately both unsuccessfully.

Csaba and Marcos, I have indeed not tried centrifuging my antibody tubes!I think we filtered them once through a .2 micron syringe filter, but I do not recall this to help much. I could be wrong though. Will try this, I suppose you use the supernatant?

Marcos, we could possibly still go down with the concentration. Thanks for your suggestion. Also, so far I have only been using 4% bovine serum albumin as blocking buffer but also tried two different vials there. My reasoning for not using serum from the species the antibody was made in is that we are not seeing unspecific staining (at least in my opinion) of blood vessels or anything like this. The more I think about this, the more it seems to me that somehow the antigen gets pulled out of the cell and then form clots. 

For permeabilizing, we have used TritonX100, Tween20, and also tried DMSO. Any more ideas? Could freezing the retinas prior to sectioning cause this?

If you have some pathology department around ask them for commercial antibody diluent (DAKO, Zytomed, CellMarque, Ventana etc). Try to impregnate your sections with it instead of blocking with tertiary serum. Some sera can even kill the immune reactivity by Fc' capture. This phenomenon can lead to precipitation like you see. I think that is what you see but I am not sure in your case. Have you tried cry or paraffin sections?

Anyway keep us updated.

Best and good luck

Ivan

Yes, centrifuge at max. speed at your regular bench centrifuge for 5 mins and use the supernatant.  

Do you freeze your samples before doing thick sections on vibratome? Is that necessary? I know that if not done properly, freezing can lead to the formation of water crystals in your samples, damaging cell membranes and creating some artifacts. In order to cryoprotect you can put your fixed samples in 30% Sucrose in Phosphate Buffer to minimize the formation of these crystals. You should let your tissue in this solution until it sinks to the bottom of the tube, i.e. losing enough water to get isotonic with the solution. After that you freeze your samples.

Marcos, it is not absolutely necessary to freeze the retinas prior to them. The idea with the sucrose is good though, I remember doing that with brain sections before and that worked quite well.

Ivan, I have not tried cryotome or paraffin sections simply because I'm trained on a vibratome and old habits die hard :) but I do want to try them.

Anyways, thank you all for your help, I appreciate the tips and got a couple of ideas for my next runs. I'll keep you posted

Interesting discussion. As you report it Felix, you have not to freeze your sample as you will not use cryocut, neither sucrose 30% as this is dedicated for frozen tissues.

Be careful when you use triton X-100, it's good for antigen retrieval but if the period (and high concentration) are higher, it could affect antigenicity.

Test your secondary antibody alone to test if the conglomerates you have are not due to it.

Good luck

One of the good things about a vibratome is that it requires almost no tissue processing prior the sectioning. Every extra step in your tissue preparation adds an intrinsic chance of generating artifacts. I would start skipping the freezing part.

good luck!

For me you have problems with fixation before the cut. Please check reagents used for this fixation.

Quick update: 

I did not freeze the samples last time, and did everything else according to our standard protocol. Unfortunately, the sections still look like above, if not worse. 

Immunostains with other antibodies I'm using all look good, so I am starting to think that the noise we are seeing is really specific to our antigen

You are right. In this case you can note the code of the antibody and  contact the manufacturer to change to change it or at least to sent you a sample to test it.