Hi, I do not have experience with these antibodies specifically, but I do have plenty of IF experience. If it is the immunofluorescence specifically you are having issues with, it makes me think it is an issue with your secondary antibody and not antigen retrieval. Have you checked your secondary antibody for specificity towards your primary? We also have better results with Tris-EDTA buffer for antigen retrieval. Another issue someone else had in our lab is that they forgot to melt the paraffin before staining, etc. What is your protocol? I would be happy to look over it and give suggestions.

Hi Skylar King. Thank you for your answer. I would also think that the secondary Ab was the problem, but it is hard to believe that since the DAB staining works quite well with a high dilution of the primary antibody. I have tested secondary antibodies from at least three vendors for HRP-based protocols. For streptavidin-based protocols, I have used only one secondary, and I am planning to test others. I also use Tris-EDTA buffer for antigen retrieval and I always melt the paraffin before staining. I have also tried melting the paraffin for 20 min and then depparafinize the slides with xylene-ethanol-water cycles.

Briefly, my HRP-based protocol is: Deparaffinization > antigen retrieval > resting in wash buffer (Tris + Tween 20) > peroxidase quenching > 3x/5 min washing (wash buffer) > blocking > primary antibody (1h at RT or overnight at 4-5ºC) > washing > secondary antibody (ready to use, or diluted 1/100, 1 h at RT) > washing > Tyramide Alexa Fluor dye (5-10 min at RT).

My brief streptavidin-based protocol is: Deparaffinization > antigen retrieval > resting in wash buffer (Tris + Tween 20) > non-serum blocking buffer (1 h/RT) > quick wash > avidin blocking (15 min/RT) > washing > biotin blocking (15 min/RT) > washing > primary antibody (1h at RT or overnight at 4-5ºC) > washing > biotinylated secondary antibody (diluted 1/100, 1h at RT) > washing > streptavidin-conjugated Alexa Fluor dye (diluted 1/100, 30 min/RT).

Have you tried just a fluorophore conjugated secondary and not an HRP or Strep-conjugated? I only use an HRP for immunohistochemistry and for fluorescence I use a conjugated- fluorophore secondary (But I also do not know the specificity of your primary antibody). We generally only use Strep-conjugated for FISH experiments, PLA or ELISA. A cheaper option that I have had great luck with in the past is Jackson ImmunoResearch Secondary Antibodies (1:200 dilution once reconstituted because it comes in powder). I have used them in C. elegans, zebra fish and mice. My suggestion would be to order a secondary here, or even any secondary that is not HRP or Strep specific and try the protocol skipping the HRP or Strep conjugation. You could also do a dot blot assay to see if your secondary is even binding to your primary, so you are not wasting your samples.

Hi Skylar King,

I have tried a fluorophore-conjugated secondary, but the signal is quite low and inconclusive. This is why I have been using signal amplification protocols.

As I said, I don't really believe that my secondary is not binding to the primary, otherwise the assay would not work on bright field IHC. But the western blot assay is actually a good idea. Thank you very much for your advice!

Good luck! Hope you get some answers!

Update:

I have tried a TSA protocol again using a GATA-3 primary antibody by Abcam (ab199428), a horse anti-rabbit IgG polymer (part of the ImmPress kit by Vector Labs) and a Cy5 dye by Biotium (equivalent to CF647, cat. no. 96022). Apparently it worked well. Since GATA-3 is highly expressed in my test tissue sample, a dye dimmer than Cy3/AF555 or Cy2/AF488 could be a better option.

I am now trying to get RORgt to work and will hopefully come back with new updates soon.

Cheers.