Sample preparation/fixation glutaraldehyde

Harvested samples should be immediately prepared for the SEM by fixation in a 0.5% osmium tetroxide solution in the dark for 1 h. If the sample is collected in the field, store in buffered 3% glutaraldehyde solution (pH 7.2) until samples can be fixed. 

Those interested in using immunohistochemistry to study apoptosis need to consider the method of tissue preparation and fixation. Since many antibody epitopes do not survive formalin/glutaraldehyde fixation or paraffin embedding, investigators should determine under what conditions the antibody of interest will work prior to sample collection. There are antibodies that will successfully bind to formalin-fixed, paraffin-embedded material, but if the investigator is unsure, fresh snap-frozen samples can be used to optimize conditions for success since freezing generally will not alter epitopes. 

Transmission electron microscopy (TEM) is important to monitor cell-free sperm decondensation and nuclear formation. To prepare specimens for TEM, incubation mixture aliquots were fixed for 45 min in 2.5% (v/v) paraformaldehyde, 3.1% (v/v) glutaraldehyde, 0.02% (w/v) picric acid in 30 mAf NaHP04, pH 7.5. After fixation, samples were embedded in 2% (w/v) low-gelling-temperature agarose and postfixed for 15 min in 1% (w/v) OSO4. Samples may be dehydrated either in ethanol and propylene oxide or acetone and embedded for sectioning in Spurr s low-viscosity epoxy resin (Spurr, 1%9). Before examination, sections of about 70-nm thickness should be stained, e.g., with uranyl acetate and Reynolds lead citrate (Re)molds, 1%3). 

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