Any optimised approaches to apply to general procedures. Does anyone have tips/recommendations for fixation composition [e.g. additives to Karnovsky's] or embedding etc. of mouse sciatic nerve for TEM?
You can use Karnovsky as a fixative or do situ fixation with glutaraldehyde
I would do a half strength Karnovsky's if you are immersion fixing (2% PF 2% Ga), I would also use phosphate or HEPES buffer, keep the osmolarity at 300 mOsmols. I would add 3 mM MgCl2 for membrane tonicity. For nerve, following the buffer (with 3% sucrose) rinse, I would use potassium ferrocyanide reduced osmium (1% osmium 0.8% K4Fe(CN)6, followed by UA en bloc staining after rinse. After ethanol dehydration, propylene oxide or acetone transition, infiltrate in 1:1 resin with catalyst: transition solvent overnight. Next day cure at 60c after 3 changes of fresh resin. We like to rock then vacuum infiltrate (15 psi) then final rock before curing for 2 days at 60C. All pretty standard info. Good luck.
Thank you both Maria and Michael. Thanks for taking the time for a detailed response, Ill give it a go. Corey
I agree with Michael. Though a 0.1M Phosphate buffer would keep the molality of the solution low enough.
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