Ab5506

After washing sections (4-μm thick) from tissue embedded in paraffin block with phosphate-buffered saline (PBS, pH 7.4) and 3% dehydroxide solution for 5 min, sections were preincubated with blocking solution (Invitrogen, Carlsbad, CA, United States) for 30 min before being incubated with the anti-ICC (ab5506, Abcam, Cambridge, United Kingdom). After the sections were incubated for 90 min with the primary antibodies, they were washed with PBS again before being incubated with secondary antibody (Alexa Fluor 488 goat anti-rabbit antibody, Invitrogen, CA, United States) for 90 min at 24 °C. After rewashing with PBS, the specimens were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) and mounted. The immunostained tissues were evaluated with confocal laser scanning microscopy (LSM 5 EXCITER; Carl Zeiss, Jena, Germany), using a C-Apochromat objective lens (× 40). Image analysis was completed with LSM software (version 3.98, Carl Zeiss, Jena, Germany). Intensity of fluorescence in the DM group was compared with that in the control group and expressed as a percentage.

Cardiac TCs were seeded on a polylysine-coated 1 cm² glass cover slip and inoculated in culture medium for 48 h at 37°C with 5% CO₂, and the culture medium was replenished after 24 h. Cells were then fixed in 4% paraformaldehyde for 15 min, washed twice with PBS and blocked with PBS containing 5% BSA for 30 min. This was followed by primary antibody probing of CD117 (ab5506, Abcam, USA), CD34 (ab8158, Abcam) and vimentin (ab8978, Abcam) at 4°C overnight. Appropriate fluorophore-conjugated secondary antibodies were used to visualize the expression in immunofluorescent cell images that were captured by a TCS SP2 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Fixed testicular tissues from 3 to 7 ages of month buffalo testes were dehydrated, and then embedded in paraffin and sectioned 5 μm thick using standard procedures. Sections were processed through xylene and ethanol into water, stained with hematoxylin and eosin, or used for immunohistochemistry. These sections were washed by PBS-Tween-20 for 3 min, and exposed 3% H₂O₂-methanol for 30 min. All sections were placed into sodium citrate-hydrochloric acid buffer solution (pH 6.0), and were heated three times (6 min each time). After heated, sections were washed by PBS-tween-20 for 3 to 5 min, blocked with 5% FBS in PBS for 1 hour at room temperature, and incubated with primary antibody for overnight at 4°C, including anti-Oct4 (Abcam, ab18976, 1:100), anti-THY-1 (Abcam, ab3105, 1:100), anti-PGP9.5 (Abcam, ab10404, 1:50) and anti-c-kit (Abcam, ab5506, 1:100) respectively. After washing three times with PBS-tween-20 for 5 min each, these sections were incubated with 3% H₂O₂ for 10 min, washed with PBS-tween-20 for 3 min. These sections were exposed for 45 min at room temperature to secondary antibody (HRP-conjugated goat anti-rabbit IgG, Abcam, ab672, 1:500), washed with PBS-tween-20 as above. The sections were also stained with DBA substrate kit (Vector Laboratories, Burlington, ON, Canada) according to the manufacture’s instructions.