Fixation with Glutaraldehyde
Materials
vol (for 500 ml) of stock mg
1. 137 mM NaCl 34.25 ml 2 M
5 mM KCl 0.83 ml 3 M
1.1 mM Na2HPO4 5.5 ml 100 mM
0.4 mM KH2PO4 2 ml 100 mM
2 mM MgCl2 10 ml 100 mM
2 mM EGTA 10 ml 100 mM
5 mM PIPES 25 ml 100 mM
5.5 mM glucose 495
pH 6.1 (cytoskeleton buffer), warm.
2. Glutaraldehyde, store in tightly-sealed containers at 4oC.
3. Cytoskeleton buffer with 0.3% (w/v) Triton-X 100, 0.5% glutaraldehyde, 30 ml. Triton X-100 should be pipeted slowly with a p-200 pipetman and a trimmed tip. Wipe the outside surface of the pipet tip before adding to the buffer. Pump up and down several times to rinse out the inside surface. Mix in a fixative bottle, cap tightly, and warm in a water bath.
4. Cytoskeleton buffer with 1% glutaraldehyde, 30 ml. Mix in a fixative bottle, cap tightly, and warm in a water bath.
5. Cytoskeleton buffer with 0.5 mg/ml NaBH4, 30 ml. Prepare fresh before use in a fixation box. Bubbles should be visible. Store NaBH4 desiccated in a tightly-sealed container.
6. Phosphate buffered saline, 100 ml, warm.
7. Fixation boxes/dished.
Procedure
1. Rinse coverslip 2x with warm phosphate buffered saline.
2. Fix 1 min in buffer 3. Agitate periodically.
3. Rinse 2x with cytoskeleton buffer.
4. Fix 15 min in buffer 4. Agitate periodically.
5. Rinse 2x with cytoskeleton buffer.
6. Treat 5 min in NaBH4. Agitate periodically.
7. Rinse 2x with cytoskeleton buffer.
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