Ls005273

Mice were euthanized by cervical dislocation, and whole retinas were removed under long-wavelength illumination (630 nm, 800 μW/cm², FND/FG, Ushio, Cypress, CA). To aid in the removal of vitreous membrane from the retinal surface, we incubated retinas in a solution containing collagenase (LS005273, Worthington Biochemical, Lakewood, NJ), hyaluronidase (LS002592, Worthington Biochemical, Lakewood, NJ), and carbogen-saturated Ames’ medium (US Biologic, Memphis, TN) for 10 mins on a rocker as described by [44 ]. Afterwards, retinas were rinsed within fresh Ames’ medium and placed into physiological chamber mounted on an upright microscope (BX50, Olympus, Center Valley, PA). Retinas were maintained in the dark at 32 °C (TC-344B; Warner Instruments), and constantly perfused (2 mL/min) with carbogen-saturated NaHCO₃-buffered (22.6 mM) Ames’ medium plus 20 mM glucose (Osm 290, pH 7.4). For RGC intracellular filling and patch-clamp recordings, we used fire-polished borosilicate glass pipettes containing (in mM) 125 K-gluconate,10 KCl, 10 HEPES, 10 EGTA, 4 Mg-ATP, 1 Na-GTP, and 0.1 ALEXA 555 (Invitrogen, Carlsbad CA; Osm 285, pH 7.35). RGC light responses were evoked using full-field light flashes generated by a light-emitting diode (365 nm, 300 μW/cm2, 3-s duration; Roithner Lasertechnik) delivered through a shutter in the microscope condenser [29 (link), 34 (link)].

Posterior subcutaneous adipose tissue (gluteal + inguinal) or the brown part of the interscapular fat was dissected, minced with scissors, and digested with 0.1 w.u./ml purified collagenase (LS005273, Worthington Biochemical, Lakewood, NJ, USA) and 2.4 U/ml Neutral Protease (LS02104, Worthington Biochemical) in Hank’s balanced salt solution (HBSS, Sigma-Aldrich, Munich, Germany) containing 4 mM calcium chloride and 0.05 mg/ml DNase I (1284932001, Roche Diagnostics, Grenzach-Wyhlen, Germany) for 50 min at 37°C in a shaker. The suspensions were strained through a 300 μ mesh (4-1411, Neolab, Heidelberg, Germany). Floating mature adipocytes and SVF were separated by centrifugation at 145 × g for 10 min at 20°C. SVF cells were washed, and centrifuged at 300 × g for 5 min at 20°C.