Goat anti tbx5

Cells were collected and lysed with RIPA buffer supplemented with protease inhibitor and phenylmethanesulfonyl fluoride (PMSF) for 15 minutes on ice. The lysate was then centrifuged at 16000 xg for 10 minutes and supernatant was added with 4x SDS loading buffer (Bio-Rad) and boiled for 5 minutes at 95 °C. Cleared lysate was run on a 4–15% gradient SDS-PAGE gel (Bio-Rad) and proteins were transferred to nitrocellulose membranes. After blocking with 5% milk, proteins were probed with primary antibodies: rabbit anti-GFP (Invitrogen, 1:500), rabbit anti-Mef2c (Abcam, 1:1000), goat anti-Gata4 (Santa Cruz Biotechnology 1:200), and goat anti-Tbx5 (Santa Cruz Biotechnology, 1:200). The target proteins were detected by chemiluminescence (ECL, Thermo Scientific). The membranes were then stripped with stripping buffer (Sigma) for 12 minutes and re-probed with mouse anti-β-actin (Santa Cruz Biotechnology, 1:1000) as the loading control. Quantification was performed with Image J.

Standard histological procedures were used (Roux et al., 2015 (link)). Heart from Hoxb1^GoF;Mef2c-Cre and littermate controls were fixed in neutral-buffered 4% paraformaldehyde in PBS, rinsed, dehydrated, paraffin-embedded and tissue sections cut at 8 μm. Sections were stained with Harris’ hematoxylin and eosin (H and E) (Sigma). For immunostaining embryos from Hoxb1^-/- or Hoxb1^GoF;Mef2c-Cre and littermate controls were fixed at 4°C for 20 min in 4% paraformaldehyde, rinsed in PBS, equilibrated to 15% sucrose and embedded in O.C.T. Cryo-sections were cut at 12 μm, washed in PBS and pre-incubated in blocking solution (1%BSA, 1% Serum, 0.2% Tween20 in PBS). Primary antibodies were applied overnight at 4°C, followed by secondary detection using Alexa Fluor conjugated (Molecular Probes) secondary antibodies. Sections were photographed using an AxioImager Z2 microscope (Zeiss) and photographed with an Axiocam digital camera (Zen 2011, Zeiss).
The following primary antibodies were used in this study: rabbit anti-Hoxb1 (Covance; 1/200), rabbit anti-GFP (Life Technologies; 1/500), mouse anti-αactinin (sigma; 1/500), mouse anti-MF-20 (DHSB; 1/100), Rabbit anti-Caspase3 (Cell Signaling Technology, 1/300), rabbit anti-phospho-Histone H3 (Millipore; 1/400), and mouse anti-Islet1 (DSHB; 1/100), rabbit anti-Tbx1 (Isbio Ls-C31179, 1/100), goat anti-Tbx5 (Santa Cruz sc-7866, 1/250).

Ten micrometers frozen sagittal sections of mouse hind limbs at the age of 1 day were subjected to fluorescence immunohistochemistry analysis using different primary antibodies (goat anti-EGR2 1:1,000, goat anti-TBX5 1:100, goat anti-DLX5 1:100, goat anti-GKLF 1:100, goat anti-SRY 1:100, goat anti-COL10A1 1:100) (Santa Cruz Biotechnology, CA, USA). Sections washed three times with phosphate buffer (PBS, PH 7.4), and permeabilized with ice-cold 0.3% Triton X-100 for 10 min at room temperature (RT), and blocked in PBS containing 5% goat serum albumin (BSA) for 30 min at RT. The sections were incubated overnight at 4°C with above primary antigen. Non-immune goat IgG was used as a negative control. After washing with the PBS (Tris Buffered Saline with 0.1% Tween-20), the slides were further incubated with Alexa Fluor 488 – conjugated Affinipure Rabbit Anti- Goat IgG (1:200, Santa Cruz) for 1 h at room temperature. Nuclear counterstaining was performed with DAPI for 10 min at RT. Immunofluorescence images were acquired using A Zeiss fluorescence microscope with 20×.