Rabbit anti sm mhc

Mouse ureters were fixed in 4% paraformaldehyde for 30 min and embedded in Optimal Cutting Temperature compound (Tissue-Tek). Frozen samples for immunohistochemistry were cut into 6 µm in thickness. Primary antibodies used in this study were as follows: rabbit anti-Smad4 (1∶100, Millipore), rabbit anti-Sox9 (1∶300, Millipore), mouse anti-αSMA (1∶100, Sigma), rabbit anti-SM-MHC (1∶100, Biomedical Technologies), rabbit anti-Uroplakin (1∶100, a generous gift from Dr. Tung-Tien Sun, NYU) [22] (link). Alexa Flour 488 or 594 conjugated secondary antibodies (1∶500; Invitrogen) were applied to detect the corresponding primary antibodies. Section RNA in situ hybridization was carried out on 12-µm cryosections with methods described previously [23] , [24] (link).

Carotid arteries and aortas were harvested and prefixed in 4% paraformaldehyde for 1 h at room temperature, and incubated in 30% sucrose for cryopreservation. Tissues were embedded in optimal cutting temperature (OCT) compound (Tissue-Tek; Sakura Finetek) and cryosectioned (10 μm) using a cryostat (NX70, Thermo Fisher Scientific). Sections were stained with hematoxylin and eosin for routine histology. For immunohistochemistry, sections were permeabilized in phosphate buffered saline (PBS) containing 0.1% Triton, and blocked in PBS with 5% normal serum. Then, sections were stained with mouse anti-αSMA (1A4) (Sigma Aldrich), rabbit anti-SM-MHC (Biomedical Technologies/ Alpha Aesar BT-562), goat PDGFRa (R&D Systems AF1062), rat anti-CD31 (MEC 13.3), rat anti-CD34 (RAM34), rat anti- CD45 (30-F11), rat anti-Sca1 (E13-161.7) (BD Biosciences), mouse anti-VE-cadherin (F-8), and mouse anti-CD68 (KP1) (Santa Cruz Biotechnology) used at 1:100. Secondary reagents were Alexa Fluor 647 conjugated antibodies from Invitrogen used at 1:200. Slides were covered with Vectashield containing DAPI (Vector Laboratories) and viewed under an LSM 710 laser scanning microscope (Zeiss) or Axio Imager.Z2 (Zeiss). Images were analyzed for quantification using Fiji Image J. At least three mice were used for each experiment (except N = 2 in Fig. 2 and Figure S4), and at least 10 sections were examined.