Alexafluor 568

The following antibodies were used for immunofluorescence: mouse monoclonal anti-human/mouse proinsulin biotinylated antibody (1:200; R&D Systems (Minneapolis, MN, USA), #BAM13361), mouse monoclonal anti-calnexin antibody (1:100; Novus Biologicals (Englewood, CO, USA), #NB300-518), rabbit polyclonal anti-GM130 antibody (1:200; Novus Biologicals, #NBP2-53420), guinea pig polyclonal anti-insulin antibody (1:50; GeneTex (Zeeland, MI, USA), #GTX27842). Secondary antibodies were from ThermoFisher Scientific: anti-streptavidin AlexaFluor-568 (1:200; #S11226), AlexaFluor-488 (1:200; #A11001 and #A11073) and AlexaFluor-568 (1:200; #A10042).
The antibodies used for immunoblotting were the following: mouse monoclonal anti-actin antibody (C4) (1:500), rat monoclonal anti-GRP78/Bip (1:250) and mouse monoclonal anti-vinculin (1:1000) from Santa Cruz Biotechnology (Dallas, TX, USA), rabbit polyclonal anti-Phospho-eIF2α (1:500), rabbit polyclonal anti-eIF2α (1:500) and mouse monoclonal anti-Chop (clone L63F7) (1:200) from Cell Signaling (Danvers, MA, USA), rabbit polyclonal anti-Atf4 (1:1000) from GeneTex. Secondary antibodies conjugated with HRP were from Biolegend (anti-mouse and anti-rabbit) and from Santa Cruz Biotechnology (anti-rat). Standard blocking conditions (5% milk in TBS-T) were used throughout, except when anti-P-eIF2α antibody was used, and 1% BSA in TBS-T was utilized.

Immunofluorescence analyses for tight junction proteins of the mice intestinal tissue were performed were performed with 4mm-thick frozen sections. Slides were fixed with acetone and blocked with 5% bovine serum albumin then incubated with antibodies against ZO-1 (Abcam, USA) or Occludin (Abcam, USA) overnight at 4°C. Subsequently, the sections were washed with PBS for 5 min three times and incubated with Alexa Fluor 488 (Santa Cruz Biotechnology, Inc) at room temperature in the dark for 60 min. Nuclear staining was achieved by 4’, 6-diamidino-2-phenylindol (DAPI). Double immunofluorescence analyses for macrophage of liver tissue were performed as shown above. Slides were fixed with acetone and blocked with 5% bovine serum albumin then incubated with antibodies against F4/80 (ab16911, Abcam, Cambridge, MA, USA) and RIP3 (Abcam, USA), or F4/80 (Abcam, USA) and MLKL (Abcam, USA), further incubated with Alexa Fluor 488 (Santa Cruz Biotechnology, Inc) and Alexa Fluor 568 antibody (Santa Cruz Biotechnology, Inc). DAPI was lastly applied to dye the nucleus. Fluorescence photographs were obtained under fluorescence microscope DM5000 B (Leika, Germany).