.. Immunohistochemistry and microscopy Embryo (E) timing was based upon identification of
coital plugs (equal to E0.5). Immunohistochemical methods were as described (Rogers and Gahring 2012; Rogers et al. 2012). Embryos were fixed in PBS/2% paraformaldehyde/5% sucrose, cryoprotected with sucrose in PBS to a final of 30%, embedded and sectioned using a Microm EM550 microtome. The 12-μm sections were mounted on glass slides, blocked, and permeabilized with 1% deoxycholate and 0.2% Triton X-100 in PBS, and then Inhibitors,research,lifescience,medical incubated overnight at 4°C with the appropriate primary antibodies. After washing, sections were incubated with secondary antibodies conjugated to fluorescent markers (Jackson ImmunoResearch, West Grove, Pennsylvania) for 1 h at room temperature. The sections were again washed, and mounted in prolog gold antifade reagent (Invitrogen, Grand Island, New York; “type”:”entrez-protein”,”attrs”:”text”:”P36930″,”term_id”:”2506565″,”term_text”:”P36930″P36930) Inhibitors,research,lifescience,medical and cover-slipped before being photographed using fluorescence microscopy (Rogers et al. 2012). Images
were collected using a Microfire 24-bit CCD camera (Optronics, Goleta, California) and imported into Photoshop C2 for Selleckchem FG 4592 preparation of figures. The antibodies used were commercially obtained. These were anti-calcitonin gene-related protein (CGRP; rabbit; 1:30; Chemicon/Millipore, Inhibitors,research,lifescience,medical Temecula, Californa AB5920), anti-GFP (chicken; 1:800, Aves Labs, Tigard, Oregon GFP-1020), anti-HA (rabbit; Inhibitors,research,lifescience,medical 1:200; HA.11 Covance, Princeton, New Jersey PRB-101P), anti-peripherin (rabbit; 1:100; Abcam, Cambridge, Massachusetts #1530), anti-S100beta (rabbit; 1:100; Abcam ab868), rabbit anti-beta-III tubulin (TUJ1; 1:3000; Covance MMS-435P). Detection of GFP
offers superior sensitivity that Inhibitors,research,lifescience,medical is well over background fluorescence (Fig. 1B and C). For this study, some inconsistent signal detection or autoflourescence was occasionally observed and these sites identified in the individual figures. We find the expression of GFP and HA are similar, although anti-HA expression is detected predominantly on the surface of cells identified by anti-GFP expression (Fig. 1D). Results The expression of α7 exhibits distinct spatiotemporal patterning in developing cochlear structures. Previously, Thymidine kinase we demonstrated the earliest expression of α7 in the developing embryo to be in rhombomeres 3 and 5 of the E9.0 embryo (Rogers et al. 2012). Thus, we initiated studies of α7GFP staining at this time. From E9.5 through approximately E12.5, the otic and cochlear structures did not express detectable α7GFP (Fig. 2A and not shown, see Rogers et al. 2012). The earliest detected expression of α7GFP in the cochlear structures was at E13.5 in cells of the spiral prominence (SP; Fig. 2B). The SP retains α7GFP expression throughout embryonic and post-natal development (see below). By E14.5 (Fig.