Hi, I want to study the membrane using SEM, I am having some problems with cell fixation. I am using PFA and glutaraldehyde , and one lasts step with HDMS ( hexamethyldisilazane).
But I think the membranes are broken due to fixation process.
Thanks
Hello,
Cells on coverslips in a well?
(Work under the hood!)
You can just fix with 1% glutaraldehyde for 30 minutes
washes with water
(optional after washing, 1% osmium then washes)
Dehydration with increasing concentration of ethanol (something like 50%, 70%, 95%, 2x100%), 10 minutes bathes
2 bathes with HMDS, 10 minutes bathes
Remove the coverslips from the well and let dry on filter paper under the hood overnight (you can speed up by placing in dry warm chamber)
Metal sputtering
I grow the cells in suspension and then I use polylysine to attach the cell in the coverslip.
I thinl the problem is during the fixation with PFA and glutaraldehide or maybe HDMS could be damaged the cell.
I read about a vacum method but i dont know if this protocol is allowed using inmunogold labelling
Do you use home-made PFA or is it ready-to-use formalin?
One of my colleague doing AFM saw lipid extraction in few seconds after adding formalin during live imaging. When he used my home-made PFA, lipid extraction was minor after 15-20 minutes fixation.
Just fix PFA and look at the same time with the microscope you have in the cell culture room. You may see huge lipid droplets bubbling from cells.
In EM i am using a lot GA only, but of course it is not very compatible with immuno(gold)labelling.
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