Yu-Li Wang Lab,University of massachusetts Medical School(UMASS)

http://ylwang.umassmed.edu/protocol/cfs/glutar.htm

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.

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.