Cellsens dimension imaging software version 2.1

Formalin-fixed tissue sections (5 microns) were deparaffinized in a series of xylenes and graded alcohols, followed by washing in de-ionized water. After unmasking the antigens using sodium citrate buffer, pH 6.0, sections were washed in PBS and incubated in a humidified chamber for 1 hour at room temperature in 5% normal blocking serum in PBS. After removal of the blocking serum, HRP-conjugated primary antibodies (Santa Cruz Biotechnology, Inc. Dallas, TX, USA) diluted in PBS with 5% normal blocking serum (2 µg/mL) was added to the sections prior to incubation overnight at 4°C. After washing in PBS, a mixture of hydrogen peroxide (H₂O₂) and 3,3′-diaminobenzidine (DAB) diluted in 0.1 M Tris-HCl pH 7.6 was added to the section until development of color. The sections were washed in deionized H₂O and counterstained with hematoxylin followed by dehydration in ethanol and xylene, then mounted with SignalStain mounting medium (Cell Signaling, Danvers, MA, USA). Images were captured at 40X magnification using the Olympus BX53 microscope, DP72 digital camera, and CellSens Dimension imaging software (version 2.1) from Olympus America, Inc. (Center Valley, PA, USA), attached to a HP Z44 computer.

After 24 hours of incubation in 4% PFA, the retinas were washed in tris maleate buffer (pH 7.2) on ice prior to incubation in ADPase incubation medium containing 3.0 mM lead nitrate and 6.0 mM magnesium chloride (Sigma Chemical Co., St. Louis, MO) in tris maleate buffer (pH 7.2). After incubation, the retinas were washed with tris maleate buffer prior to addition of diluted ammonium sulfide (Fisher Scientific, Silver Spring, MD, USA) for 1 min. The retinas were washed again in tris maleate buffer and flat-mounted on a microscope slide with PBS and glycerin. All images were captured using an Olympus BX53 microscope, DP72 digital camera, and CellSens Dimension imaging software (version 2.1) from Olympus America, Inc. (Center Valley, PA, USA), attached to a HP Z44 computer.

GFAP and isolectin B4 double staining of the retinal flatmounts were conducted as previously described [56 (link),57 (link),58 (link)]. Briefly, retinal flatmounts were washed in ice-cold PBS/Triton X-100 (TXPBS), fixed in methanol, followed by permeabilization and blocking in PermBlock (PBS + 0.3% Triton X-100 + 0.2% bovine serum albumin) in 5% goat serum for 1 hour. After washing in TXPBS, flatmounts were incubated with rabbit GFAP primary antibody (Cell Signaling Technologies, Danvers, MA, USA) overnight at 4°C. Following several washes with TXPBS, the flatmounts were incubated with Alexa Fluor 488 goat anti-rabbit fluorescent secondary antibodies, and Alexa Fluor 594 Isolectin B4 (ThermoFisher Sci/Life Technologies, Grand Island, NY, USA) overnight at 4 °C. The flatmounts were washed with TXPBS and mounted on slides with prolong anti-fade fluorescent mounting media and imaged at 20X magnification using the Olympus BX53 microscope, DP72 digital camera, and CellSens Dimension imaging software (version 2.1) from Olympus America, Inc. (Center Valley, PA, USA), attached to an HP Z44 computer.