I've been staining 6um sections of FFPE human spinal cord tissue with fluorophore conjugated primary antibodies for multiplex immunofluorescence experiments and have noticed extensive non-specific binding of what appears to be myelin (photo attached in which AF488 is NeuN). As you can see, the specific grey matter staining is scant and the non-specific white matter staining is blasting. This only occurs in spinal cord (not brain from the same cases) and is much less visible if performing indirect immunofluorescence of the same antibody target. I've tried multiple different antigen retrieval protocols (citrate in microwave, citrate in autoclave, and Tris-EDTA in autoclave). I've also tried blocking with serum (donkey and horse), BSA, serum+BSA. I also used the blocking agent as diluent with different concentrations of detergent. All to no avail. Any insight on what else I can try?
try treating sections with fresh sodium borohydrate (0.1% weight/volume) or 0.1M Glycine solution in PBS. Both pre-treatment reduce free aldehyde, which nonspecifically bind your antibodies. Google it and you find protocols and papers. This phenomenon is typical in tissue sections, where the tissue was fixed for prolonged period of time (over-fixed). Reduce the length of time you use to fix the tissue; when using PFA, increase the length of PBS wash and wash several times (sometimes overnight at 4C in a large volume) to remove the fixative from tissue before it is embedded. PFA fixation is partially reversible. Critically, user FRESH PFA, purchased in brown vials and freshly diluted. PFA (especially diluted to 4% from 16% stock in brown vials) must be kept for not more than ~2weeks in the fridge and protected from light, you can prepare a large batch of 4% and then freeze at -20 in small aliquots, this works great. Using old PFA solution and/or PFA, which was exposed to light is one reason for seeing such unspecific signal since PFA is gradually converted to Glutaraldehyde, especially at warmer temp and in bottles not protected by light (fixing with Glut is not reversible and adds to the problem you see). Last, use fixatives other than PFA such as Bouin fixative. Using the 2 quick fixes (Glycine or sodiuym borohydrate) will be a "quick fix" to your problem but changing the ways of doing IHC will help to eliminate the problem. good luck
If that doesn't work, you could try using 0.1% Sudan Black B (right before mounting, just google "sudan black autofluorescence" for a protocol). I have had success using this approach to quench autofluorescence in the 480-490 channel from several different tissue types.
Thanks, Igor O. Nasonkin! I'll try using Glycine.
Thanks, Melissa Bemrose! I already use Sudan Black B in my protocol but it seems that the fluorescence is coming from non-specific binding to myelin rather than autofluorescence from lipofuscin or other sources.
oh! I should have read your question more closely. Best of luck to you!
Update: I tried adding glycine to my blocking agent as per protocols online to no avail. The non-specific myelin staining is still there only in the spinal cord (not the brain of the same patient cases) and only with fluorophore-conjugated antibodies.
Any other suggestions on things I can try?
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