Alexa fluor 555

Eighty animals (20 R6/2 treated with vehicle, 20 R6/2 treated with Olaparib, 20 vehicle and 20 Olaparib-treated Wild type mice) were transcardially perfused under deep anesthesia with saline solution containing 0.01 mL heparin, brains were removed and cut in half. Tissue sectioning was performed on a sliding frozen microtome at 40 μm thickness. All brain sections were processed for single label EM-48 ubiquitin (1:500, Chemicon, Temecula, CA); NLRP3 (1:200, Abcam, Novus Biologicals, Italy); Calbindin (1:500, Abcam, Novus Biologicals, Italy); Parvalbumin (1:200, Chemicon International, Inc., Temecula, CA, USA); Calretinin (1:200, Chemicon International, Inc., Temecula, CA, USA), and GFAP (1:600 polyclonal anti-GFAP, Millipore, Italy). We used Alexa Fluor 488 and Alexa Fluor 555 (Immunological Sciences, Italy) as secondary antibodies. Brain sections from the same level of the bregma, were mounted on gelatin-coated slices and cover slipped with Gel-Mount. Samples were examined with the support of confocal laser scanner microscopy (Zeiss LSM 800); images were acquired and subsequently analyzed to quantify the immunofluorescence intensity of Calbindin, NLRP3, Parvalbumin, Calretinin, and GFAP-positive cells. To evaluate pyroptosis, peroxidase-antiperoxidase diaminobenzidine tetrahydrochloride single-label immunohistochemistry for caspase-1 was performed [23 (link)].

After the stimulation, CHO cells were washed in PBS 1×, blocked for 1 h in 5% normal donkey serum and then incubated overnight at 4 °C with a rabbit polyclonal antibody against PKR2. Cells were then washed with PBS 1× and incubated for 1 h at room temperature with secondary anti-species IgG antibodies coupled to Alexa Fluor-555 (Immunological Sciences, Rome, Italy). The cell nuclei were stained with DAPI. The fluorescence signal was recorded using an Eclipse E600 fluorescence microscope (Nikon Instruments, Tokyo, Japan) connected to a high-resolution digital camera (Nikon Digital Sight DSU1) with 64-bit NIS-Elements BR 3.2 software.