Abstract
Indirect immunofluorescence (IF) microscopy is a frequently used method to determine intracellular protein localization. It is especially useful for low abundance proteins, for example the GATA-factors (Gln3, Gat1) which activate nitrogen catabolite repression (NCR)-sensitive transcription. Limiting nitrogen or treating cells with Tor pathway inhibitor, rapamycin, elicits nuclear GATA-factor localization and increased NCR-sensitive transcription, whereas excess nitrogen restricts these proteins to the cytoplasm and decreases transcription. The initial step of the IF procedure is formalin-fixation that quenches cellular activity and fixes protein locations via cross-linking. We find that under some conditions, formalin itself can influence GATA-factor localization. With low formalin (0.8% or 1.6%), Gat1-Myc(13) became more nuclear, and with higher concentrations (5.6%), it became more cytoplasmic. Gln3-Myc(13) localization, on the other hand, did not respond to low formalin, but became more cytoplasmic at the higher concentration. Interestingly, the high concentration of formalin had no demonstrable effect when the GATA factors were completely nuclear, i.e. after rapamycin (Gat1-Myc(13)) or Msx (Gln3-Myc(13)) treatment. These effects are most likely elicited by polyoxymethylene glycols, which significantly increase the osmolarity of the medium (0.5-2). We suggest that varying degrees of osmotic stress and transcription factor movement in response to it can occur after the beginning of fixation but before proteins become immobilized.