Gluteraldehyde fixation of cells

Glutaraldehyde fixation (for optimum cytoskeletal preservation)and immunofluorescence:Stock solutions:PBSFix 1 :Fix + Triton X-100:Antibody buffer 2 :1X BRB800.25% glutaraldehyde1X BRB800.25% glutaraldehyde0.1% Triton X-100PBS2% BSA0.1% Triton X-100Mowiol: 0.1 M Tris-HCl pH 8.525 % glycerol10 % Mowiol 4-88 (Hoechst) 3ProtocolWarm solutions to room temperature 4 .Wash coverslip in PBS for a few seconds.Drain coverslip as much as possible by holding the edge on a paper towel. Remove anymajor drops of PBS.Quickly dip into Fix (in a Coplin jar).Incubate for 20-30 seconds.Transfer coverslip to Fix + Triton X-100.Incubate for 10 minutes.1 Both fixatives may be prepared ahead of time and kept frozen in 10-ml aliquots.2 Goes bad fairly quickly. For storage add 0.02% sodium azide and keep refrigerated.3 Dissolves really slow. Mix for several hours. Centrifuge out any residual undissolved material. Store inaliquots at -20ºC.4 For cells from warm-blooded animals 37ºC is even better.

Transfer coverslip to PBS 5 .Place coverslips sample side up on Parafilm (do not let them dry!)Cover with a fat drop of freshly prepared 0.2% sodium borohydride in PBS 6 .Incubate 20-30 minutes changing the borohydride solution two or three times.Wash twice in PBS.Incubate coverslips for 10 minutes in antibody buffer to block unspecific binding.In the meantime, dilute primary antibodies in antibody buffer (rat anti-tubulin 1:500;mouse anti-vinculin 1:250). For big square coverslips, about 50 µl antibody solution percoverslip should be sufficient.Incubate coverslips with primary antibodies for 30 minutes.Wash three times with PBS.In the meantime, dilute secondary antibodies in antibody buffer (1:500), add Alexa488phalloidin (1:500) and Hoechst dye (1:500).Incubate coverslips with secondary antibodies for 20 minutes.Wash three times with PBS.Drain most of the PBS and mount in Mowiol (about 10 µl for a big square coverslip). Tryto avoid bubbles as much as possible.Let Mowiol set for 30 min at 37ºC or overnight at room temperature.5 Coverslips can be stored like this at 4ºC for next day immunofluorescence.6 When glutaraldehyde reacts with amino groups it forms a fluorescent component which needs to bereduced directly by hydrogen generated in situ by hydrolysis of sodium borohydride. If it does not bubble itis no good! Glycine or Tris will not work.