Coolsnap hq

After fixation in 4% paraformaldehyde, brains were incubated in 30% sucrose, frozen in Jung tissue medium (Leica, Nanterre, France) and sectioned using a cryostat. Sagittal sections (10 μm) were washed in PBS with 0.25% gelatin and 25% Triton and incubated at 4°C for 24 h with rabbit anti-ionized binding molecule adaptor 1 (Iba1) (Wako, Osaka, Japan) and rabbit anti-pPKR_Thr446 (Abcam, Cambridge, UK) and at room temperature for 2 h with secondary antibodies donkey anti-rabbit Cy3 (Jackson Laboratory, Bar Harbor, Maine, USA). Standard epifluorescence images were acquired on a Leica DMRD microscope using a high resolution camera (Coolsnap HQ). The Metamorph software (Roper Scientific, Sarasota, FL, USA) was used for image acquisition. All quantitative image analyses were performed by using NIH ImageJ software, as previously described35 (link). Quantification was limited to the areas corresponding to the cortex and hippocampus. DAPI and the cyanine 3 pictures were both background corrected using the rolling ball method. A threshold was then chosen using the ‘Auto threshold' function of ImageJ. Cells were subsequently evaluated by defining a region of interest and by running the ‘Analyze particles' imageJ function. Afterward, quantification of positive cells was performed using the Colocalization plug-in.