Goat anti gfp

Sections were washed 3 × 20 minutes in PGT (0.45% Gelatin and 0.25% Triton in 1× PBS) at room temperature. Next, primary antibodies (1:1,000 goat anti-GFP from Novus Biologicals (Littleton, Colorado, United States of America) or 1:10,000 rabbit anti-RFP from VWR (Rockland)) were incubated with PGT overnight at 4°C. Following 10-, 20-, and 40-minute washes in PGT, sections were incubated with secondary antibodies (1:1,000 donkey anti-goat coupled to A488 or 1:1,000 donkey anti-rabbit coupled to A555, both from Molecular Probes (Eugene, Oregon, United States)) in PGT at room temperature for 1 hour. Sections were subsequently washed for 10 and 20 minutes in PGT and 30 minutes in PBS. Sections were then mounted on glass slides and permanently coverslipped with Fluoromount that contained DAPI.

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.

Transcardial perfusion with 4% paraformaldehyde (PFA) was performed on the mice for fixation. The brains of E17.5, P0, P2, P4, P7, P14, P30 mice were dissected and post-fixed at 4°C with 4% PFA for 2h to overnight, depending on the size of brains. The brains were dehydrated in 30% sucrose and embedded in OCT compound. Cryosections were cut at 14-μm, or 80-μm thickness with a Cryostat (HM505E, Microm, Germany). Immunostaining was performed with standard protocols: brain sections were incubated overnight with primary antibodies at 4°C and incubated with appropriate fluorescent secondary antibodies for 2h at room temperature. The following primary antibodies were used: Goat anti-GFP (1:1000, Novus Biologicals); Mouse anti-FOXP1 (1:125, Abcam); Rabbit anti-Tbr2 (1:300, Abcam); Rabbit anti-Tbr1 (1:200, Abcam); Mouse anti-Satb2 (1:100, Santa Cruz); Mouse anti-Nestin (1:200, Abcam); Rabbit anti-CDP (1:50, Santa Cruz); Rat anti-Ctip2 (1:500, Abcam); Mouse anti-phospho Histone H3(1:300, Abcam); Mouse anti-β-III-tubulin (1:500, Promega).
Immunofluorescence images were obtained using either Olympus BX41 or Zeiss LSM 710 confocal microscope. The morphology of neurons in the cortex or culture was traced and analyzed using Neurolucida software (MBF Bioscience).