Mouse anti tg2 cub 7402

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.

Sections of human liver were obtained from formalin-fixed and paraffin-embedded blocks of retrospective/archival samples (i.e., patient selection and liver biopsy were not for the purpose of the study). Replicate blocks, no longer required to be maintained on file within the local pathology archive, were used. Six cases of NAFLD and six cases of normal control tissue were selected; the presence of a hepatitis B or hepatitis C infection, or of alcoholic liver disease were considered as exclusion criteria.
For immunohistochemistry, deparaffinized and rehydrated sections were immersed in 10 mM sodium citrate, pH 6.0, and microwaved for antigen retrieval. Samples were incubated with mouse anti-TG2 (CUB 7402, Thermo Scientific, Rockford, IL, USA), for 1 h at room temperature. Reaction was visualized using a streptavidin–biotin–immunoperoxidase system with DAB (Biogenex, San Ramon, CA) as chromogen substrates. Negative control staining was performed by omitting the primary antibody. Sections were counterstained in Mayer’s acid hemalum.