For John Vournakis, Biology Department
Materials:
12mm round cover slips
Eagle's DMEM without serum (physiologically compatible buffer)
0.2M Na Cacodylate stock Buffer pH 7.2
2% Glutaraldehyde in Eagle's DMEM without serum
2% Glutaraldehyde in 0.1M Na Cacodylate pH 7.2 with 0.1M Sucrose
Fix ampules contain 2 ml of 70%GTA. 2%GTA = 2ml ampoule + 68ml buffer for a total of 70 mls. of soln.
Fixation Methods:
1. Replace growth media with 2% Glutaraldehyde in Eagle's DMEM without serum. Do this several times to ensure a complete transition from growth media to fixative. Fix for 0.5 hours. at room temperature.
2. Transfer cover slips to fresh vials or petri dishes containing 2% Glutaraldehyde in 0.1M Na Cacodylate pH 7.2 with 0.1M Sucrose and fix for a further 1.5 hours. at room temperature.
3. Store samples overnight in fix at 4 C, if necessary.
Dehydration, CPD, Mount and Sputter Coating:
1. Rinse in 0.1M NaCacodylate pH 7.2. Transfer cover slips to CPD holder.
2. Dehydrate in ethanol series (30%,50%,70%, 95% and 3 x in 100%, 5 mins each).
3. Critical point dry.
4. Mount cover slips on Al stubs.
5. Carbon coat, using vacuum evaporater(@100? ), and sputter coat with 100 ? AuPd.
Specimen preparation for DRY(in-house)CHITIN matrix (unused; no cells):
1. Attach Matrix to round Al mounts with spot tape. Be careful not to "stretch" filters when mounting.
2. Sputter Coat with 150 ? AuPd.
