Parvalbumin

Formononetin was obtained from Selleck Chemicals Company (Houston, TX, USA; S2299). Cisplatin was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA, Cat. No. P4394). The following primary antibodies were used in this study: myosin-VIIa (Proteus Biosciences, Ramona, CA, USA; 25–6790), parvalbumin (Abcam, UK; ab277625), cleaved caspase-3 (Cell Signaling Technology, Beverly, MA, USA; 9661S), caspase-3 (CST, 9662S), Bax (CST, 2772S), Bcl-2 (CST, 3498S), PI3K (CST, 4249S), phospho-PI3K (CST, 17366S), AKT (CST, 4691S), phospho-AKT (CST, 4060S), and Nrf2 (CST, 12721S). MitoSOX RED (Invitrogen, Eugene, Oregon, USA, M36008) and LY294002 (Selleck, S1105) were also utilized in the experimental procedures.

Retinal immunohistochemistry was performed as described by Song et al.²⁵ (link) Briefly, eyes were fixed, cryoprotected, embedded, and snap-frozen in Tissue-Tek O.C.T. compound (Sakura Finetek USA, Inc., Torrance, CA), and cryo-sectioned at 14-µm thickness. Cryo-sections were blocked in blocking buffer and incubated with anti-GFP (mouse, 1:1000;, Cell Signaling Technology, Danvers, MA), or Calbindin (mouse, 1:2000; Sigma-Aldrich, St. Louis, MO), or Parvalbumin (rabbit, 1:1000; Abcam, Cambridge, MA), or Brn3a (mouse, 1:100; EMD Millipore, Temecula, CA), or Glutamine synthetase (GS, mouse, 1:1000; EMD Millipore, Temecula, CA) primary antibodies overnight at 4°C, and then incubated with appropriate secondary antibodies either conjugated to Alexa Fluor 488 or Alexa Fluor 568 (1:1000; Invitrogen, Eugene, OR). Retinal nuclei were counterstained with DAPI and sections were mounted in Fluoro-Gel buffer (Electron Microscopy Sciences, Hatfield, PA) for imaging. The images were generated and analyzed by the Nikon C2 confocal microscope (Nikon, Tokyo, Japan) with NIS-elements AR software, and further edited using Adobe Photoshop CC (Adobe Systems Inc., San Jose, CA). The same imaging setting (without post-processing using Photoshop) was used only for comparison of retinal GFP expression between ECVM-treated and control groups (i.e. Figs. 2A, 2B, Figs. 3C, 3D, 3E, 3F).

Brain tissues were homogenized in the sample lysis buffer (62.5 mM Tris-HCl pH 6.8, 2 % SDS, 0.5 % NP-40, 5 mM EDTA) plus the protease inhibitor cocktail (Roche). Protein samples (15-30 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Memb-ranes were probed with antibodies directed against phosphotyrosine (1:1000, Millipore, Bedford, MA, USA), ErbB4 (1:1000, Santa Cruz Biotechnology). Alter-natively, immunoblots were probed with antibodies directed against excitatory and inhibitory neuronal markers GluA1 (1:500, Millipore), GluA2/3 (1:1000, Millipore), GluA4 (1:1000, Millipore), GluN1 (1:1000, Millipore), GluN2A (1:500, Millipore), GluN2B (1: 500, Millipore), GluN2C (1:500, Millipore), GluN2D (1:1000, Santa Cruz Biotechnology), GAD65/67 (1:1000, Millipore), parvalbumin (1:3000, Abcam, Cambridge UK), tyrosine hydroxylase (1:1000, Millipore) neuregulin-1 (1:300, RandD Systems, Minneapolis, MN), and β-actin (1:2000, Millipore). Immunoreactivity on membranes was detected by peroxidase-conjugated anti-immunoglobulin antibodies followed by chemiluminescence reaction (ECL kit, GE Healthcare) and film exposure. The intensity of an immunoreactive band, whose size matched the authentic molecular weight, was measured by an image processing software, GENETOOLS (Syngene, Cambridge, UK).