The 2fTGH cells were fixed in ice-cold ethanol/acetone (1:1) at −20 °C for 10 min. Samples were briefly rinsed in phosphate-buffered saline (PBS), permeabilized with 0.1 % Triton X-100 in PBS for 5 min and blocked with 1 % bovine serum albumin (BSA) and 10 % normal goat serum in PBS for 30 min. Primary antibodies, namely mouse anti-TG2 (CUB 7402, Thermo Scientific, Rockford, Ill., USA), rabbit anti-calreticulin antibody (Stressgen, Enzo Life Sciences, Farmingdale, N.Y., USA), rabbit anti-p62/SQSTM1 (MBL, Woburn, Mass., USA) and rabbit anti-Tom20 antibody (Santa Cruz Biotechnology, Dallas, Texas, USA), were incubated for 1 h with the cells at room temperature. After being washed, the cells were incubated with Alexa488- or Alexa594-fluorochrome-coupled secondary antibodies directed against rabbit or mouse (Molecular Probes, Life Technologies, Grand Island, N.Y., USA).
Coverslips were mounted in SlowFade-Anti-Fade (Invitrogen, Life Technologies). Fluorescence was analyzed with a TCS SP2 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). Digital images obtained separately in both channels through a 63× objective (zoom factor 2×) were acquired with Leica Confocal Software.
Coverslips were mounted in SlowFade-Anti-Fade (Invitrogen, Life Technologies). Fluorescence was analyzed with a TCS SP2 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). Digital images obtained separately in both channels through a 63× objective (zoom factor 2×) were acquired with Leica Confocal Software.