Fitc conjugated goat anti mouse igg sc 2010

To confirm that AAV-2 viral particles were successfully delivered into the retinal cells of the experimental animals, mice were sacrificed by cervical dislocation. The eyes were enucleated and washed with PBS and prefixed in 4% paraformaldehyde for 10 min. The iris and lens were removed, and the retina was carefully separated from the sclera and fixed in ice-cold methanol for 20 min. After washing, tissues were blocked with 1% bovine serum albumin (BSA) at RT for 90 min. The tissues were then incubated with mouse anti-GFP antibodies (sc-9996, 1:100, Santa Cruz Biotechnology, Germany) overnight at 4 °C. On the next day, the tissues were washed with PBS and incubated with FITC-conjugated goat anti-mouse IgG (sc-2010, 1:200, Santa Cruz Biotechnology, Germany) for 2 h at RT. Finally, the tissues were visualized by an Axiophot Zeiss fluorescence microscope. Isolectin B4 staining was performed to reveal the vessels. Whole-mounted retinas were incubated overnight at 4 °C with isolectin B4 (lectin from Bandeiraea simplicifolia (L2895, Sigma-Aldrich, Germany), 1:200). Images were acquired using a Leica TCS SPE confocal microscope. AngioTool software was used to quantify the total vessel length and vessels area. The vessels of each retina were observed in three different fields for each studied animal, and the mean value was calculated and compared between all groups.

The eyes of the animals were enucleated and the retinas were isolated. The retinal samples were then placed in 10% formalin for 2 days. Following fixation, the retina was incubated in trypsin (3% in sodium phosphate buffer) for ~60 min at 37°C. The vessel structures were isolated from the retinal cells by gentle rinsing in distilled water. The vascular specimens were then mounted on a slide. For immunofluorescence staining for HMGB1 and NF-κB, the vascular specimens were incubated with rabbit anti-HMGB1 (Epitomics) and mouse anti-NF-κB antibody (cat. no. MAB3026; Chemicon International, Inc., Temecula, CA, USA). For the detection of HMGB1, the vessels were incubated with FITC-conjugated polyclonal goat anti-rabbit antibody (sc-2012; Santa Cruz Biotechnology, Inc.). To detect NF-κB, the vessels were incubated with polyclonal FITC-conjugated goat anti-mouse IgG (sc-2010; Santa Cruz Biotechnology, Inc.) and detected using fluorescence microscopy (Olympus Corporation). For negative controls, the sections were incubated with non-immune serum instead of the primary antibodies.