1. To fix cells, remove from incubator, work on bench top. Wash cells 2 times with PBS, fix for 5-10 min with fixative (NBF, Bouin's, or 4% paraformaldehyde), wash 3-4 times with PBS. Store at 4_C in PBS-0.1% NaN3 (for as long as 2 years). See the following for 4% paraformaldehyde fixative.

PREPARATION OF 4%-PARAFORMALDEHYDE FIXATIVE
NOTE: This procedure should be performed under a fume hood. Paraformaldehyde produces strong fumes, and may be a reproductive toxin or carcinogen. Wear gloves.

1. Heat ~100 ml DDW to 60-70_C.

2. While heating, weigh out 4g p-formaldehyde for a conc. of 8% (2X) in 50 ml DDW. Place in a 150 ml beaker and set aside.

3. Prepare 50 ml of 2X TBS (20 mM Trizma Base, 300 mM NaCl):

a. Add ~876.6 mg NaCl to a 100 ml beaker.

b. Add 5 ml of 20X Tris Buffer, pH 7.5.

c. Add DDW to 50 ml.

d. Mix on stir plate and set aside.

4. When DDW has been heated to 60-70oC, pour ~40 ml into the beaker containing the p-formaldehyde. Mix on stir plate with continued heating.

5. After 2-3 minutes, slowly add 10N NaOH in drops until the p-form. solution clears. This should take no more than 3 or 4 drops.

6. With continued stirring, add the 50 ml of 2X TBS prepared earlier.

7. pH to 7.5 using conc. and 1N HCl. Add DDW up to 100 ml.

8. Fixative should be used at room temperature.

9. Store at 4oC up to 2 wks.

4% PARAFORMALDEHYDE FIXATION

(6-Well Plates)

1. Wash wells X2 with TBS, pH 7.5.

2. Add ~1 ml/well 4% p-form. fixative. Incubate at room temperature for 10 min.

3. Wash X1 with TBS.

3. Add ~1 ml/well 0.1% Nonidet P-40 (NP-40). Incubate at room temperature for 2 min.

4. Wash X2 with TBS.

5. Add 3-4 ml TBS-0.1% Azide to each well. Store plate at 4oC until further use.

6. Check occasionally to make sure the wells have not dried out.

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