Fixation, permeabilization, and staining of cells

The quality of immunofluorescence of cellular components (and inununocyto-chemistiy in general) often presents conflicting demands to the investigator the cellular structures should be adequately conserved yet made readily available for antibody labelling, and the reactivity of the cellular epitopes should not be affected. To achieve this, an optimal fixation and permeabilization method should be selected, with the conditions optimized for  

In this section, we will describe several procedures including fixation with paraformaldehyde, glutaraldehyde, methanol/acetone, and EGS. 

For all the protocols described below, cells should be grown on glass cover-slips (0 = 15 mm). 

Fixation of cells by paraformaldehyde is used when cell surface staining is re quired. It is also a good choice for labelling of microfilaments, although microtubules are not fixed well by it. 

Heat about 80 ml of D-PBS to 80 °C and add 3 g paraformaldehyde while stirring. Mix until clear. 

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