Anti-SOX9 Antibody AB5535

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Publication protocol

After 2 weeks of intravitreal injections and at 3, 7, and 14 days after light exposure, the rats were euthanized and transcardially perfused with saline followed by 4% paraformaldehyde; then, their eyes were enucleated. The cornea and lens were removed, and the eyecups were immersed in the same fixative for 2 h. The eyecups were cryoprotected in graded sucrose solutions (20%–30% in a phosphate buffer [PB]), embedded in an optimal cutting temperature (OCT) compound (Tissue-Tek; Ted Pella, Inc., Redding, CA), snap frozen in liquid nitrogen, and then sectioned (10 μm). Air-dried slides were incubated with a blocking buffer (phosphate buffer saline [PBS] containing 5% goat serum and 0.25% Triton X-100) at room temperature for 1 h. After washing three times in 0.01 M PBS, the sections were incubated overnight at 4 °C with the following primary antibodies: rabbit polyclonal antibody against Sox9 (1:800; Millipore, MA); mouse monoclonal antibody against glutamine synthetase (GS); (a specific Müller cell marker; 1:200; Abcam, MA, USA); and mouse monoclonal antibody against GFAP (1:1000; MAB360, Millipore, MA, USA). The sections were then rinsed three times in 0.01 M PBS and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (1:800; Invitrogen, Carlsbad, CA) at room temperature for 1 h. After three rinses with 0.01 M PBS, they were counterstained with DAPI (1:800, Sigma-Aldrich, St. Louis, MO) and examined using a photomicroscope (Leica Microsystems, Bensheim, Germany).

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