Dmrd microscope

Hemispheres from vehicle- and CQ-treated mice were stained with an enhanced Golgi-Cox staining system (superGolgi Kit, Bioenno Tech), following manufacturer instructions. Briefly, 150 µm thick brain sections were cut using a vibratome (Leica) and mounted on adhesive microscope slides. Air-dried sections were stained, cleared in xylene, coverslipped in Eukitt mounting medium, and stored in the dark until analyzed. Images from secondary dendrites of CA1 pyramidal neurons were acquired with a DMRD microscope (Leica) equipped with a DFC550 CCD camera (Leica) and a 100× oil-immersion objective (1.3 NA; Leica). A trained experimenter blind to treatment manually counted the spines.

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.

After 24 h from the induction of the oxidative stress, DRG cultures were fixed with ice-cold 4% paraformaldehyde for 5 minutes at room temperature. Cultures were stained with anti-β-tubulin III primary antibody (1:1000 Sigma, UK). Cultures were then washed and incubated with the appropriate anti-mouse secondary antibodies with Alexa Fluor-488 (1:2000 Molecular Probes, UK). Cultures were also counterstained with the fluorescent nuclear stain, 4′,6′-diamidino-2-phenylindole (Hoechst 2 μg/ml; Sigma, UK). Cells were observed at ×20 magnification using a Leica DMRD microscope (Leica, UK). Images were captured using a Hamamatsu (Hamamatsu, Japan) digital camera and HiPic32 imaging software. Adobe Photoshop 7 was used to produce micrographs.