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SEM Protocol for Fibroblast Cells grown on (in-house)CHITIN matrix

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.

Last Updated: 10/2/08