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.
I would try something different, like a 4% paraformaldehyde, 1% glutaraldehyde plus 1% or less of Acrolein in phosphate buffer. The fixative is so penetrant and immediate that there is no time for tonicity effects to change the structure. Variants of this mixture have been the best for a great variety of tissues, including marine invertebrate. It has to be used with caution in a hood.
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