Fitc conjugated rabbit anti mouse igg secondary antibody

To study the distribution and expression of Opa1 in the retinal vasculature, retinal capillary networks were subjected to immunostaining with Opa1 antibody. The retinal capillary networks were washed several times with 1× PBS and subjected briefly to ice-cold methanol, followed by additional PBS washes. Then, the RTDs were exposed to a 2% BSA solution diluted in 1× PBS for 15 min at room temperature to block non-specific antibody binding. Following blocking, the RTDs were subjected to a primary antibody solution containing mouse monoclonal Opa1 antibody (1:200 in 2% BSA-PBS solution, Catalog #sc-393296, Santa Cruz Biotechnology, Dallas, TX, USA) and incubated overnight at 4 °C in a moist chamber. The following day, RTDs were washed in PBS and incubated at room temperature with FITC-conjugated rabbit anti-mouse IgG secondary antibody (1:100 in 2% BSA-PBS Solution, Catalog #315-097-003, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After three PBS washes, RTDs were counterstained with DAPI, and mounted in SlowFade Diamond Antifade Mountant reagent (SlowFade Diamond; Molecular Probes, Eugene, OR, USA). Digital images were captured, and Opa1 immunostaining was quantified from 10 random representative fields from each RTD, with equal exposure time for all compared panels for each antigen.

To study the expression and distribution of Drp1 in the retinal capillary networks, retinal trypsin digestion (RTD) preparations [36 (link)] were subjected to immunostaining with Drp1 antibody. The RTD preparations were washed several times with 1× PBS and subjected briefly to ice-cold methanol, followed by additional PBS washes. Then, the RTDs were exposed to a 2% BSA solution diluted in 1× PBS for 15 min at room temperature to block non-specific antibody binding. Following blocking, the RTDs were subjected to a primary antibody solution containing mouse monoclonal Drp1 antibody (1:200 in 2% BSA-PBS solution, Catalog #sc-271583, Santa Cruz Biotechnology, Dallas, TX, USA) and incubated overnight at 4 °C in a moist chamber. After the overnight incubation, the RTDs were washed in PBS and incubated at room temperature with FITC-conjugated rabbit anti–mouse IgG secondary antibody (1:100 in 2% BSA-PBS Solution, Jackson for 1 h). After three PBS washes, RTDs were counterstained with DAPI, and mounted in SlowFade Diamond Antifade Mountant reagent (SlowFade Diamond; Molecular Probes, Eugene, OR, USA). Digital images were captured, and relative Drp1 immunofluorescence was quantified using the NIH Image J software from 10 random representative fields from each RTD.