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[egerecske]

Hi all,

I have no experience in electron microscopy. If I have PFA-fixed brain tissues, can I use them for electron microscopy?
In the literature, I found that PFA+glutaraldehyde fixation is needed, can this glutaraldehyde fixation be given later to the already PFA fixated tissue? What is the reason of using different concentrations (2%PFA/2%glutaraldehyde, or 4%PFA/0,1%glutaraldehyde)? What does the glutaraldehide to the sample?

[victorius]

Hi,

Here is a fixation protocol that worked for my brains:

-BEWARE: osmium tetroxide, uranyl acetate and the embedding resins are all very nasty, dangerous, toxic stuff. Use a fume hood and read all warnings!!

AFter PFA fixation, cut very small pieces of tissue (~1 mm diameter) and:

Postfix in 2% glutaraldehyde in cacodylate buffer (CB) 0.1 M for 1-12 hs.
Rinse in CB, 10 min, 2 times
Fix in 1% Osmium tetroxide in CB x 1 hr.
Rinse in physiological saline x 10 min, 2 times
Soak in 2% uranyl acetate in 10% ethanol for 1 h. (for contrast)
Wash in 50% Ethanol x 15 min.
Wash in 50% Ethanol x 15 min.
Wash in 70% Ethanol x 15 min.
Wash in 70% Ethanol x 15 min.
Wash in 95% Ethanol x 15 min.
Wash in 95% Ethanol x 15 min.
Wash in 100% Ethanol x 15 min.
Wash in 100% Ethanol x 15 min. (You can stop here and keep tissue in the fridge)
Wash in 100% Acetone 15 min.
Wash in 100% Acetone 15 min.
Infiltrate with 1/2 acetone + 1/2 resin (e.g. Spurr) x 2 hs
Infiltrate with 1/3 acetone + 2/3 resin x 2 hs
Infiltrate with 100% resin x 14 hs
Put samples in BEEM capsules with fresh 100% resin, bake at 72ºC overnight.

Good luck!

v/