Goat anti mouse fab fragment

Mice were euthanized using 60mg/kg sodium pentobarbital and tracheostomized with an angiocatheter. Mouse heart and lungs were removed together, perfused with saline, inflated through the catheter with 10% neutral buffered formalin, fixed in 10% neutral buffered formalin, and embedded in paraffin blocks(15 (link)). For immunofluorescence, tissue sections were deparaffinized and antigens were retrieved by boiling in citrate buffer for 30 minutes. Endogenous mouse IgG was blocked with goat-anti mouse Fab fragment (Jackson ImmunoResearch, 115-007-003). Fluorescent images were captured using a Zeiss Axioplan epifluorescence microscope. Primary antibodies used were: BD Biosciences mouse anti-αE-cat (#610193), Sigma mouse anti-desmin (D1033), and rabbit anti-αT-cat (polyclonal #952, kindly provided by Frans van Roy, Ghent University, Belgium). For quantification, the intensity of each cardiomyocyte junction was measured using ImageJ from exposure-matched images. H&E staining was performed by the Northwestern University Mouse Pathology Core Facility.

Similar to hematoxylin and eosin staining, we performed a series of tissue incubations in xylene and ethanol to remove paraffin and hydrate the tissue. Following this step, we performed antigen retrieval in sodium citrate (pH6.0) and permeabilization in Triton X-100 (VWR). We than performed a series of washing steps to remove the detergent. We then performed two blocking steps, each lasting one hour. The first blocking step was 10% BSA in PBS. After two brief washing steps in PBS, we than incubated the slides in 40 μg/ml of goat anti-mouse Fab fragment in PBS (Jackson ImmunoResearch Laboratories, 115-007-003). Primary antibodies were incubated for 18–24 hours at 4°C. Primary antibodies included TP63 (Santa Cruz, 4A4, sc-8431), Keratin 6 (Covance, PRB-169P), Keratin 14 (Novocastra, NCL-L-LL002), IRF6 (Sigma-Aldrich, SAB2102995), Keratin 1 (NCL-CK1, Novocastra), Loricrin (PRB-145P, Covance), Ki-67 (ab15580, Abcam), GRHL3 (a kind gift from Dr. B. Andersen, UCI, Irvine, CA), Activated Caspase 3 (Abcam, Ab13847), KRT17 (Sigma-Aldrich, HPA000453) JAG2 (ThermoFisher, PI701287) and MMP13 (Sigma-Aldrich, HPA030636). Secondary antibodies (goat anti-mouse and goat anti-rabbit (Molecular Probes)) were incubated for 1–2 hours. Nuclei were labeled with DAPI (Invitrogen).