Alexa 488 anti goat

Temporal bones were removed after euthanasia in 4 to 8 weeks old mice and placed in 4% PFA for 1 hour, followed by decalcification for 24 to 36 hours with 120 mM EDTA. The sensory epithelium was then dissected out, permeabilized with 0.01% Triton-X and incubated for 24–48 hours with primary antibodies. Rabbit anti-MYO7A primary antibody (1:500, #25–6790, Proteus Bioscience, CA) and Goat anti-GFP (1:500, #NB100–1770, Novus) were applied for 48 hours. Secondary antibodies, Alexa-488 anti-goat and Alexa-633 anti-rabbit IgG (1:200, Invitrogen) were applied for 2–3 hours along with Alexa phalloidin 633 to label actin filaments (Invitrogen, 1:200). Images were obtained on a LSM710 and LSM800 Zeiss confocal microscope (IDDRC Imaging Core grant P30 HD18655) and processed with Zeiss LSM image viewer 4.2.

Whole embryos were fixed for 1 h in 70 % ethanol. The dorsal skin was then dissected and postfixed overnight in 4 % PFA. The samples were washed twice in PBS containing 0.2 % Triton X-100 (PBT) 30 min at 4 °C and blocked for 1 h in PBT containing 1 % BSA at RT. To perform double immunostaining, primary antibody against CD31 (at 1/150) and VEGF-R3 (at 1/50) (AF743, R&D Systems, USA) was diluted in PBT/BSA and incubated overnight at 4 °C. After three washings of 30 min in PBT at 4 °C and two at RT, the skin samples were incubated overnight at 4 °C with Alexa 488 anti-goat (1/1000 in PBT/BSA) and Alexa 546 anti-rat (1/1000) (Invitrogen, USA). After washing (as above), samples were mounted on glass plates with Aquapolymount (PolySciences Inc., USA) and observed with a Leika SP5 confocal microscope, adapted from [32 (link)].