Donkey anti rat igg h l alexa fluor 647 preadsorbed

Following intracardiac perfusion of 10 ml room-temperature PBS, mice were perfused with 10 ml of room-temperature 4% paraformaldehyde (PFA) in PBS. Cochleas were dissected and fixed for 24 hours in 4% PFA. Cochleas were decalcified in 10% EDTA in Tris-buffered saline pH 7.4 for 7 days, then sectioned at 50 μm with a vibratome (22 ). Tissues were washed in PBS, then blocked with 0.5% Triton X-100 and 5% donkey serum in PBS for 1 hour, followed by incubation with primary antibody in blocking solution overnight at 4 °C (Ly6G, 1A8 clone BioLegend cat# ab154885; 2.5 μg/ml). Sections were washed 3× in PBS, then incubated with secondary antibody in blocking solution overnight (Donkey Anti-Rat IgG H&L (Alexa Fluor 647) preadsorbed; abcam cat# ab150155, 0.5 μg/ml). Sections were washed 3×1 hour in PBS followed by treatment with Streptavidin Alexa Fluor 568 (ThermoFisher, Cat# S11226) at 10 μg/ml in PBS with 0.5% Triton X-100, 0.1 mM CaCl₂, and 10% donkey serum for 1 hour. Tissues were washed 2×1 hour in PBS with 0.2% Triton X-100 and incubated overnight with Hoescht (1 ug/ml) and Lycopersicon Esculentum lectin-488 (Vector cat# DL-1174; 200 μg/ml) in PBS. Next, sections were washed 2×1 hour with 0.2% Triton X-100 in PBS, washed 1×1 hour with PBS, post-fixed in 4% PFA for 15 minutes then washed 3×20 minutes in PBS, then mounted on slides with Vectashield mounting medium.