I have 50 micron thick sections of a rat brain which I am trying to stain with primary antibody AT8 (for p-tau). I stained at 1:500 dilution overnight followed by a 3-hour incubation with secondary antibody. Upon imaging I saw that the AT8 had stained only the top and bottom surfaces of the tissue section. I then incubated another section with 1:500 AT8 for 2 nights on a shaker, hoping it will improve tissue penetration of the antibody. But that did not help either. Note that these sections had not undergone antigen retrieval. Does anyone have any experience with IHC staining of such thick sections?
How is the tissue prepared? How long was it fixed? What is the strength of the detergent you are using you are using for staining? I have only used citrate antigen retrieval for paraffin embedded sections so I don't suspect that is an issue here. Properly permeabilized free floating 40um brain sections should stain with .1%-.2% TritonX/ 3% BSA. In my experience, Tau and even Map2 don't always look great on brain slices--- more suited for axon and dendrite staining in vitro.
Are 50 micron thick sections not thick for staining? You should try to stain with thinner sections (eg. 20 microns) or with ranging from 10 to 40 micron sections. Otherwise, permeabilization process could damage to your sections.
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