I feel like I have tried everything and wanted to reach out to you all to see if anyone has any suggestions for staining the dentate gyrus of my 40 um mouse tissue with DCX at 1:500. I have tried using H202, a pretreatment with .1 M Tris and .3% Triton, as well as .01 M Citric acid (pH 6) antigen retrieval for 10 minutes at 90 degrees. I have used different combinations of these steps as well as them individually and have an overwhelming amount of background and feel as if there is nonspecific staining occurring.
Thank you!
Hello Brittini,
I suggest you are working with fixed frozen or vibratome sections. This antibody is not suitable for this application. Abcam is very pricisely in validation of application. I have made a similar expierence with another antibody. I recommand to read the datasheet and look for which kind of application the antibody is suitable. In your case I would recommand the ab207175 (abcam) or#326003 (Synaptic Systems)
Hi, you can put the sections with citrate buffer at boiling temp. for 5 min and then at RT for another 20 min. Also could you please share your blocking buffer as well as primary & secondary antibody dilution preparation & incubation time?
Thanks
Meetu Wadhwa My blocking buffer includes .3% PBST with 5% Normal Donkey Serum for 1 hour prior to primary antibody incubation. My primary antibody prep is 1:600 DCX in blocking buffer overnight at 4C. My secondary is Donkey ant Goat 555 (1:500) plus 3% NDS in PBS for 1.5 hours at room temp.
Thank you so much!
The information that I can see on abcam (ab113435) is that this antibody is suitable IHC-P. Although IHC-P antibodies work for IHC-Fr as well, still I would suggest you to try on paraffin sections as well so that we can come to know that whether the antibody is not working or there is something wrong in protocol.
Meanwhile, I would like to suggest you some modifications that you may try as mentioned below-
Antigen retrieval - Try 10mM sodium citrate tribasic (pH-6) with 0.05% tween-20. Blocking - You may try 0.1% PBST with 5% NDS as blocking buffer for 30 min at RT.
Primary antibody - DCX in blocking buffer for 40h at 4 degree C. Sometimes, particular lot of abcam antibodies do not work at 1:500/1:1000. Have you tried to use 1:250 or 1:100 dilution?
Washing - PBS with 0.1% triton-x100.
Let me know if these modifications work or not.
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