Improved fixation for immunofluorescence microscopy using light- activated 1,3,5-triazido-2,4,6-trinitrobenzene (TTB)

A new fixation method has been developed for immunofluorescent microscopy using the photosensitive compound 1,3,5-triazido-2,4,6- trinitrobenzene (TTB). Our results show that TTB-fixed cells are well preserved morphologically and that the cellular antigens are better preserved than conventionally fixed cells. By altering one condition at a time in the TTB fixation procedure and analyzing resulting fluorescent antitubulin staining patterns in mammalian tissue culture cells, an optimal procedure was developed. Cells fixed with TTB and stained with antitubulin, antiprekeratin, anti-intermediate filament, anti-alpha-actinin, anti-myosin, antiactin, or anticlathrin were compared with cells fixed by conventional methods and stained with the same antibody. The quality of immunofluorescence images of TTB fixed cells was the same as or better than that of conventionally fixed cells. The most dramatic improvement in image quality was seen when using antiprekeratin or antitubulin. In dividing cells, particularly in metaphase, fluorescent staining with antiactin and anti-alpha-actinin was relatively excluded from the spindle. Antimyosin, on the other hand, stained the spindle and surrounding area more heavily than the subcortical region. We suggest that after TTB fixation, the immunofluorescent patterns of these contractile proteins more closely reflect their relative concentrations in living cells. The exact mechanism for fixation by TTB is not yet known. However, our studies indicated that TTB fixation was not caused by the typical fast photoinduced nitrene diradical mechanism, but rather by some slower, temperature-dependent reaction of a photoactivation product of TTB with the cell.