Glick fix

A method for staining ER and small structures which are difficult to visualise or poorly preserved with other fixation methods. This method is probably not good for cell surface staining!

 

Grow cells on coverslips, transfer to a container with cold methanol or acetone stored at —20°C and then leave for 10 minutes. The choice of solvent depends on the antigen and needs to be determined empirically.

After fixation, remove the coverslips from the solvent and air dry at room temperature.

Rehydrate the fixed cells for 30 minutes in a drop of PBS containing 0.1% n-octyl-b-D-glucopyranoside (Calbiochem) and 100 µM bis(sulfosuccinimidyl) suberate added fresh from a 10 mM stock stored at —80°C (BS3, Pierce Chemical).

Wash 3x with PBS containing 0.1% n-octyl-b-D-glucopyranoside, and then quench the residual BS3 with a drop of 0.1M ethylenediamine-HCl pH 7.5 for 15 minutes.

To remove the ethylenediamine solution wash 3x with PBS containing 0.1% n-octyl-b-D-glucopyranoside.

 

Carry out all antibody and blocking incubations in PBS containing 0.1% n-octyl-b-D-glucopyranoside and appropriate blocking agents, eg 1% milk powder and/or 1% fish skin gelatin as needed.

 

Taken from: Hammond, A.T., and B.S Glick (2000) Mol. Biol. Cell 11:3013-3030.