Cellular immunofluorescence: quantification of low abundance proteins.

Aminofluorescein can be extracted with alkaline carbonate buffer (0.5% Na2CO3 in 0.1 M NaOH) from fixed cells stained in indirect immunofluorescence by fluorescein-conjugated antibody. Fluorescence is then quantified by spectrofluorometry. A standard curve obtained by dilution of known fluorochrome allows for subsequent spectrofluorometric analysis of the extracted aminofluorescein. A saturating quantity of primary antibody should be used to determine the level of staining associated with a cellular antigen. This simple method makes it possible to quantify samples used for immunofluorescence microscopy. It can be adapted for determining the DNA content in the same samples, allowing the quantity of antigen to be equated either to DNA content or to cells plated.