Some protocol mentioned the saline perfusion for mouse brain to flush out blood is very short, say less than 2 minutes for detection of synaptic changes for electron microscopy. They say it undergo transformation, retraction or whatever if not fixed with Karnovsky fixative immediately which is not the case for IHC. Longer saline infusion flushes out the RBCs completely and it gives a better quality image. Just for observations of ultrastructural changes e.g mitochondria, ER or golgi apartus, what is the appropriate duration (in minutes) for saline infusion just before the PFA+Glutaraldehyde.
We typically perfuse mice with 10-15 ml of heparinized 0.1M phosphate buffer for 10 min, so with a flow of 1-1.5 ml per minute. We also look at spine morphology and we can spot differences in mutant mice vs wild type controls, although I don't have a comparison with a shorter wash and Karnovsky fixative (so I cannot say if we are losing something or not...). Anyway, as far as I know, the original Karnovsky fixative is not used anymore because it is highly hypertonic and today you can find modifications (diluted versions, and without the cacodylate buffer) which are not very different from what we use, 4% formaldehyde, 2.5% glutarladehyde in 0.1M phosphate buffer...
Madam in the EM lab also told that saline isn't good for perfusion of mouse brain for EM. She had used heparine injected directly in to the heart just before the PFA+GLU infusion. Anyway, thanks for sharing the detail information. It was helpful. One query- how much unit of heparine did you use in 0.1M PB?
PB is not saline, which is not good because it does not buffer and because of the Cl-. PB is phosphate buffer, obtained mixing approximately 1 part of NaH2PO4 and 4 parts of Na2HPO4, which has the double function at 0.1M concentration of buffering at pH 7 and bringing in Na+ at about 180 mM, only slightly hyper-osmolar to the cells (but total OSM 280, very close to blood OSM).
We use 0.2% w/v heparin.
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