Fixation with Glutaraldehyde

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 Na2 HPO4 5.5 ml 100 mM

0.4 mM KH2 PO4 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 4o C.

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.