Anti c kit

The testicular tissues were treated with a 4% paraformaldehyde solution for fixation and then dehydrated in different concentrations of ethanol (70%, 90%, and 100%). Following embedding in paraffin blocks, the tissues underwent sectioning and subsequent rehydration, involving xylene for a duration of 30 min, followed by 5‐min intervals of 100%, 85%, and 75% ethanol. To perform immunostaining, the sections underwent permeabilization using 0.1% (v/v) Tween‐20. Following the addition of 3% (v/v) BSA to obstruct, the sections were subjected to incubation with anti‐DEAD‐box helicase 4 (DDX4/MVH) (Abcam, cat no. ab13840, 1:100), anti‐Lin28 (Abcam, cat no. ab46020, 1:200), and anti‐c‐kit (Abcam, cat no. ab32363, 1:100) antibodies for an extended period at 4°C. On the following day, the samples were exposed to secondary antibodies at ambient temperature in the absence of light for a duration of 50 min. Additionally, they were stained with Hoechst (diluted to 1:2000; Invitrogen) for 10 min at room temperature. In order to perform quantitative analysis of cells, the quantities of MVH⁺, Lin28⁺, and c‐kit⁺ cells in each cross‐section of the testis were tallied in five randomly selected fields per section. For each group, analysis was conducted on a minimum of three sections obtained from separate experiments. All images were obtained using an IX73 microscope.

Cells were harvested with Laemlli buffer and heated for 10 minutes at 100°C. Protein concentration was estimated using bicinchoninic acid (BCA) assay kit purchased from Thermo scientific following manufacturer’s instructions. Before loading the samples on the gel, 3 μl of β-mercaptoethanol was added per 100 ul of sample and heated for 3 minutes at 100°C. Cell lysate (25 or 50 μg of protein) was loaded on 4–20% Tris-Glycine gel and separated by electrophoresis. The separated proteins were transferred to a PVDF membrane, blocked with 5% skim milk or 5% bovine serum albumin for 1 hr followed by probing with primary antibody overnight at 4°C. The blot was then incubated with HRP-conjugated secondary antibody for 1 hr and developed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). Primary antibodies used were anti-c-kit (1 in 500; abcam #32363), anti-phospho c-kit (Y703; 1 in 500; Cell Signaling #3073P), anti-AKT (1in 1,000; Cell Signaling #4691), anti-phospho AKT (Thr 308; 1 in 1,000; Cell Signaling #13038), anti-ERK 1/2 (1in 1,000; Cell Signaling #4695P), anti-phospho ERK 1/2 (1 in 1,000; Cell Signaling #4370P), and anti-α-tubulin (1 in 3,000; Sigma #T6074).

Primary antibodies were γ-H2AX (Abcam, Cambridge, UK, Anti-gamma H2A.X (S139 antibody [9F3], ab26350) or GAPDH (14C10) rabbit IgG mAb (Cell Signaling Technology, Cambridge, UK CST, 2118), anti-ckit (Abcam, ab32363), Anti-eNOS (Cell Signaling Technology, 9572), or PDGFR-b (Abcam, ab69506); anti-beta Actin (Abcam, ab8227), anti-IL6 (Abcam, ab6672), anti-phospho-Rb (Abcam, ab47763), anti-p21 (Abcam, ab109520), or anti-CD163 (Abcam, ab156769); anti-mTOR (Abcam, ab2732), anti-NFkB (Santa Cruz Biotechnologies, Inc., Dallas, TX, USA, sc8008), anti-ATM (Abcam, ab32420), or anti-GLUT1 (Abcam, ab115730); CD34 (Santa Cruz Biotechnologies, Inc., Dallas, TX, USA, sc74499). Secondary antibodies: AF488GAM (Molecular Probes, ThermoFisher Scientific, Waltham, MA, USA; Novus Biogicals, LLC, Centennial, CO, USA, A11029), AF546GAR Molecular Probes, A11035), HRP-anti-mouse IgG (BD Pharmigen^TM (BD Biosciences), San Jose, CA, USA, 7076S), and HRP-anti-rabbit IgG (BD Pharmigen^TM, 7074S).

Tissue sections was processeded by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining for assessing the apoptosis of the transplanted BMSCs, anti-von Willebrand factor (vWF, Abcam Ltd, Cambridge, UK) staining for the evaluation of angiogenesis, Masson trichrome staining to delineate the myofilament structure, as well as anti-cTNT, anti-c-kit, and anti-BrdU staining (Abcam Ltd) to identify CSCs and neonatal cardiomyocytes. Immunofluorescence of lamella of crawling CSCs was performed with anti-cTNT and anti-connexin43 (CX43) antibodies (Abcam Ltd, Cambridge, UK) to confirm cardiac differentiation. 5 non-overlapping fields in transverse sections of each animal were randomly captured under a light or confocal microscope. Image Pro Plus (IPP) 6.0 software package (IPP, Media Cybernetics, Maryland, USA) were used to determine the myocardial density (MD) and new vessels (NV) through optical density (OD) calibration.

The skin, muscle, femur, tibia, and thymus were harvested from the recipients or allografts on determined days following transplantation and processed for paraffin sections. For morphologic evaluation, sections were stained with H&E. For immunofluorescence imaging, sections were cut at 6 µm and stained with primary antibodies: anti-Osterix, anti-c-Kit, anti-CK5, anti-CK8, anti-CD11c from Abcam (Cambridge, MA) or anti-DEC205 from Santa Cruz (Dallas, TX), followed by fluorescent secondary antibodies. DAPI (Sigma-Aldrich) was used to stain the nucleus. The slides were evaluated in a blinded fashion.

The tissue sections were cut into slices, deparaffinized, rehydrated, antigen-retrieved, stained, and then the fixed sections were stained with anti-CD31 (Abcam, Cambridge, UK), anti-c-Kit (Abcam), anti-HSP47 (Proteintech, Wuhan, China), and anti-α-SMA (Proteintech). All slices were analyzed and photographed by microscope (Leica, Germany). Microscopic angiogenesis was evaluated.