I have a problem between fixation and permeabilization of a sperm sample,
1. After Live/Dead staining, I wash my 100 uL sperm sample (25x106 sperm/mL) with 900 ul of D-PBS and centrifuge for 5 min 600g
2. After that, I resuspend the pellet with 100 uL of Formalin and incubate it for 15 min (at room temperature)
3. Then, I add approximately 900 uL of D-PBS to wash (centrifuge 5 min 600g)
4. The next step, I resuspend the pellet of 100 uL of Triton X 0.1% to permeabilize it, for 10 minutes (at room temperature protect from the light)
So, my problem occurs when I have to resuspend with 900 uL of D-PBS to wash the Triton. After centrifugation I observe agglutination of sperm (spots in suspension) and if I put 10 uL on the slide, all the sperm are agglutinated.
Does someone have any suggestion? I have tried different protocols and any of them solved my problem
thank you
Are you preparing the sperm for FACS? if not, you can fix the sperms on the slide and then proceed to the rest of the protocol. If you need the sperm cells in suspension, have you tried to fix it with ice cold acetone:methanol (1:1)? So you don't need extra permeabilization step... Although I am not sure if it is compatible with your live/dead staining.
Out of curiosity, which specie is your sperm from? I did similar protocols with bovine sperm and didn't have any problem.
Are you using BSA, PVA or another protein source in your PBS? Sperm often stick to each other or to slides when there is no protein source in the media. Another option is to try penicillamine (see Leahy 2016, Penicillamine prevents ram sperm agglutination in media that support capacitation http://www.reproduction-online.org.ezproxy1.library.usyd.edu.au/content/151/2/167.full.pdf+html), but this only works for some causes of agglutination.
I agree with what Marcia said, depends on your purpose of the staining (for FACS or for IFA), solutions can be different.
For IFA, after your live/dead staining, spot your sperm suspension onto super frost or silane-coated slides so sperm can attach to the slide better. Air dry (RT), then fix, you will not see the agglutination due to your fixation.
For FACS, you indeed can add PVA or PVP into your medium to prevent the adhesion of sperm cells.
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