Triton-Ethanol Fix.

 

Reagents.

 

TRIS-buffered Saline (TBS) pH 7.4

          Stock A.  200 mM TRIS Base. 

          Stock B.  200 mM HCl. 

          For 1 L TBS: 100 mL stock A,  44 mL Stock B.  9 g NaCl. dH2O to 1 Liter.

          Adjust to pH 7.4; store refrigerated.

 

 

Fixative

          Add 120 microliters of TX-100 to 100 mL 35% ETOH.  Store 2-6 C; keep on ice while using.  (Use wide tip pipeter).

 

 

DAPI: 1 microgram per mL.  \

 

Procedure.

 

1) Chill cells on ice.  Spin cells, aspirate supernatant.

 

2) Add 5 mLs of ice cold fix, keep on ice 10-30 minutes.  Other steps are at room temp.  (Ice is to preserve microtubules, you may not need that).

 

3) Spin, aspirate, resuspend in TBS.  (Wash 2 X). 

 

4) Stain/ mount in DAPI-vectashield.