Figure 2.

Effect of fixations, protein extractions, and dehydration of samples on protein yield. GLU/PFA fixed samples (A) lysed with RIPA buffer (0.01% SDS) on ice or (B) lysed with RIPA buffer (2% SDS) and incubations at 100 °C/20 min and 60 °C/2 h: 1) Unfixed lysates, 2) 4% PFA alone, 3) GLU (0.05%)/PFA, 4) GLU (0.01%)/PFA, 5) GLU (0.005%)/PFA. C) Percent of cells with TNTs fixed with GLU/PFA, 4% PFA alone, PFA/3 mM DTBP, PFA/5 mM DTBP, or PFA/10 mM DTBP. Graph shows means (± SEM); **p = 0.00529; *p = 0.05507 and “ns” is not significant. D) PFA/5 mM DTBP fixed cells were plated on MMI dishes and ii) cut by LCM 3 h after i) delineation of the ROI and iii) isolated subsequently. No cellular changes were observed during the LCM isolation. E) PFA/DTBP fixed samples were lysed with RIPA buffer (2% SDS and 100 mM DTT) and incubations at 37 °C/30 min, 100 °C/20 min, and 60 °C/2 h; 1) Unfixed lysates, 2) 4% PFA alone, 3) PFA/3 mM DTBP, 4) PFA/5 mM DTBP, 5) PFA/10 mM DTBP. F) PFA/5 mM DTBP fixed samples extracted and loaded as in gel E; 1) cells were fixed and lysed the same day; 2) cells were fixed and kept in PBS for 24 h before lysing; 3) cells were fixed and kept dry for 24 h before lysing; 4) cells were fixed and kept in PBS for 72 h before lysing; 5) cells were fixed and kept dry for 72 h before lysing. Stars indicate protein bands with decreased intensity. Gels are representative of three independent experiments.