Yu-Li Wang Lab,University of massachusetts Medical School(UMASS)
http://ylwang.umassmed.edu/protocol/cfs/glutar.htm
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.
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.