Hello) We are trying to visualise mitochondria in rat kidney cortex with immunoflurescence. It seems that formaldehyde fixation (via transcardial perfusion) results in degradation of mitochondrial reticulum. We have observed fine preserved mitochondrial morphlogy using electron microsopy. Is it possible to use the same fixation protocol (2,5% glataraldehyde on ice) for immunoflorescence?
Gluteraldehyde gives non-specific fluorescence with Hoechst33342, and to some extent with Alexa-488 even after long washes. Methanol fixation doesn't have such issues in my hands at least. I use methanol stored at -20 and fix the cells in plate/slides for overnight in -20. Note:the next PBS wash should have 0.1% BSA in it to keep cells in the plate/slides.
GA almost always gives unacceptable levels of autofluorescence that is impossible to remove... but as Goodwin pointed out, its in the blue and green channels... if your dye is red, go for it... Otherwise if you are desperate, you can try methacrylic acid N-hydroxy succinimidyl ester (MA-NHS)... see http://www.nature.com/nmeth/journal/v13/n6/full/nmeth.3833.html. It shows much lower autofluorescence...
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