Rabbit anti p eif2α

Double IF staining was performed as described (22 (link)). Briefly, brain tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm. After dewaxing, the sections were permeabilized in Immunostaining Permeable Buffer (Beyotime). After three washes in PBS, the sections were blocked in Immunostaining Blocking Buffer (Beyotime) for 1 h at RT and incubated with primary antibodies at 4°C overnight: rabbit anti-p-PERK (Abcam), rabbit anti-p-eIF2α (Abcam), mouse anti-NeuN (Abcam). After three washes in PBS, the sections were incubated with donkey anti-rabbit IgG Alexa Fluor 488 (Invitrogen) and donkey anti-mouse IgG Alexa Fluor 555 (Invitrogen), for 1 h at RT. Finally, counterstaining was carried out with DAPI and a U-RFL-T fluorescence microscope (OLYMPUS, Japan) was utilized for analysis.

HeLa cells were seeded in a 12-well cluster (10⁵ cells/well) and, after overnight incubation, transfected with the indicated plasmids (500 ng well; 250 ng/plasmid) using Lipofectamine2000 (Invitrogen). At 24 hr post transfection, cells were either left untreated or treated with 500 μM sodium arsenite (NaAsO₂, Riedel-de Haën) diluted in DMEM for 30 min at 37°C. Cells were detached with trypsin, washed once PBS, and fixed in PBS, 3.7% PFA for 10 min. To stain for p-eIF2α, cells were permeabilized in ice-cold MeOH for 10min, washed twice in FACS buffer (PBS + 0.02% Na-azide and 1% BSA) and subsequently incubated with the primary antibody rabbit anti-p-eIF2α (1:100, Abcam) in FACS buffer for 45 min. The samples were washed twice in FACS buffer and incubated in the secondary antibody goat anti-rabbit Alexa 594 (1:100, Invitrogen) in FACS buffer for 45 min. Cells were washed twice with FACS buffer and stored at 4°C. Fluorescence intensity was recorded with a CytoFLEX LX flow cytometer (Beckman Coulter) and the data were analyzed by FlowJo v10 software (BD Biosciences). Samples were gated for live single cell populations and then gated for negative, low (L) and high (H) RFP expressing cells. For bar graphs [H/L] ratios were normalized with the ratio calculated for the relevant wildtype protein set at 100%.