After a classical Ficoll, I spread PBMC (suspended in PBS1X) on Poly-L-Lysin pre-treated and positively charged glass slides with cytospin (200rpm during 5 min). Then I fix them with PFA 4% and cold methanol 100% before immunofluorescence.
Their nuclei look like those of polymorphonuclear cells or are broken (with "streaming DAPI").
Is this distortion known with cytospin (even so I reduced the speed and time of cytospin) ?
Try to fix with only 100% of methanol. PFA (fixative) consist sodium and formic acid which usually causes some shrinkage and/or distortion of the specimen. This might caused the distortion of the nucleus. Most of the staining (e.g: Wright's staining) for thin blood smear (obtained using blood smear or cytospin) requires methanol as the fixative agent.
All the best.
http://www.med-chem.com/pages/lab_procedures/pdf/wrights_one-step_stain.pdf
http://research.nhm.org/pdfs/1505/1505.pdf
http://www.virusys.com/documents/Fixation%20and%20Tissue%20Processing.pdf
I don't think that fixation would cause the kind of nuclear changes you describe, it is more likely to be a function of handling or the cytospin.
After cytospin, let slides dry 30 minutes to 1 hour at air and fix them.
Can you stores the cytospined slides without fixation at -20 degrees and fix as and when required. If not can you fix with methanol and then store them. If so what is the optimum storage temperature?
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