Calbindin

Sections were blocked with 2.5% normal horse serum and incubated overnight at 4 °C in primary antibodies as follows: Egr-1 and phosphorylated c-Jun N-terminal kinase (p-JNK) from Cell Signaling, aquaporin-1 (AQP1) from Santa Cruz Biotechnology (Dallas, TX, USA), and Calbindin from Abcam (Cambridge, MA, USA). After washing, the sections were incubated with Alexa Fluor488-conjugated and/or Alexa Fluor594-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA, USA). Fluorescence was visualized using a Fluoview 1000 IX-81 confocal microscope (Olympus). The fluorescence intensity of p-JNK was analyzed using Image J software, Version 1.52a (NIH, Bethesda, MD, USA).

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)].

Transduced mouse retinas were dissected and fixed in 4% paraformaldehyde for 30 min at room temperature. The retinas were incubated in PBS with 1% Triton X-100, 0.5% Tween 20 for 1 h at room temperature and in 10% normal goat serum for 1 h at room temperature and then incubated overnight at 4 °C with primary antibody: Brn3a (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rhodopsin (1:500; Abcam, Cambridge, UK), PKCα (1:500; Abcam), calbindin (1:500; Abcam) and glutamine synthetase (GS) (1:500; Abcam) in blocking buffer. Secondary anti-rabbit IgG and anti-mouse IgG conjugated with Alexa TM594 and Alexa TM555, respectively (1:1,000; Molecular Probes), were applied for 1 h at room temperature. The retinas then were flat-mounted and the sections mounted on slide glass.