Hey everyone, i am trying here to stain mouse spinal cord with parvalbumin(mouse origin), from swant, dilution 1:5000 with alexa fluor 647 or 633. The problem is that that the staining is very faint and it doesnt even look similiar to what it was expected. I tried to go down with dilution but still the same. Any ideas why?
Protocol is in fixed tissue (frozen sections) washing 3 times with PBS then put blocking buffer for 1h (0.5% Triton X-PBS and 10% serum), add primary antibody(0.3% Triton and 2.5% serum) leave overnight at 4 degrees then wash 3 times PBS, add secondary (AF647) and NT both in 1:500 dilution, leave overnight, wash 3 times PBS, coverslip with vectashield and confocal imaging.
I have heard anecdotally that hardset Vectashield significantly quenches far red antibodies. I would try mounting in Fluoromount G or Prolong Gold instead. You could try to quickly test if it is the Vectashield by cover-slipping with PBS, take a few images, then mount with Vectashield and reimage to see if there's a difference. Also the 1:5,000 working dilution for the data sheet was probably determined for HRP-DAB straining based on their images, and you may need to use a much higher concentration of primary to get the same signal/noise fluorescently without enzymatic amplification (such as TSA). I don't have hands on knowledge with this particular ab though so I can't say for sure. Hope this helps.
I was thinking about doing a signal enhancement with HRP or biotin indeed or try it free floating. If nothing works then i will check also for the Vectashield. Thank you very much for your suggestions and help.
parvalbumin immunolabelling tends to degrade over time and therefore if you kept samples in freezers too long (especially if a -20) there is very little to be seen; it is recommendable, at any rate, to do double-ON incubation. Do not forget that immunolabelling may look very different from genetically-labelled neurons. We do immunostaining for PV in spinal cord routinely in my lab and we never had any problem. We can send the protocol over if needed, of you can drop by.
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