I grow my neurons on glass surface. Their axons so delicate that I cannot remove the media entirely. When I refeed them, I remove half the media and replace it with fresh media containing double the concentration of growth factors.
Growing their axons is no problem, however if I want to perform stain them with for example, a fluorescent actin stain, I'll need to aspirate all media to add the fixative, wash, permeabilize etc. before adding the stain.
Are there any techniques that allow me to fix and permeablize my cells without aspirating all the media?
Hi Martin,
I guess fixing the cells by using the same procedure as during media exchange would be ok, just replace half the media with formaldehyde or glutaraldehyde fixative. Either use double the concentration of fixative or change the fixative (1x concentration) a couple of times to reach the desired final concentration. For permeabilization I would suggest to include Triton X-100, saponin, or Tween-20 during the final incubation step only.
Best regards and good luck with your experiments,
Christian
You can simply add an equal volume of fixative without first removing half the culture medium. That way, you'd be even faster. Also, I recommend keeping the neurons on a heating plate (35C) and adding pre-warmed (35C) fixative. After fixation you can continue with your labelling procedure at room temperature as usual. Good luck...
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