Pk 7800

Fixed spermatogenic cells were washed with PBS and permeabilized with 0.1% Triton X-100 (Sigma, T8532) in PBS containing 2% BSA (Fraction V, Sigma, A8022). Then, cells were incubated -away from light- with different markers alone or combined: a primary antibody against α-tubulin (1:50, MP Biomedicals, 691251) to localize the manchette, secondary antibody conjugated with biotin (pan-specific antibody, Vector, PK7800) and avidin-fluorescein complex (Vector, SA5001); antibody against α-actin conjugated with Cy3 (3 μg/ml, Sigma, C6198); Cholera toxin subunit β conjugated with alexa fluor 594 (5 μg/ml, MP Biomedicals, C22842) or with FITC (Sigma, C1655) in order to identify GM1 sphingolipid, a component of lipid rafts; Propidium iodide (1 μg/ml, Sigma, P4864) was applied to identify nuclear material. After washing, cells were mounted with fluoroshield (Sigma, F6182) and examined using inverted microscope NIKON TE2000.

Immunohistochemistry was performed by following the manufacturer’s instructions for vectastain universal kit, peroxidase, RTU (PK-7800, Vector Laboratories, Burlingame, CA, USA). Briefly, paraffin-embedded sections (5 μm) of the aorta were deparaffinized and prepared for antigen retrieval followed by quenching of endogenous peroxidase activity by using 3% H₂O₂. For blocking, sections were incubated with 2.5% horse serum for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4 °C against SM22-α (1:1500), RUNX2 (1:300), OSP (1:100), CD63 (1:50) and AnX2 (1:50). Subsequently, sections were incubated with biotinylated secondary antibodies and a streptavidin peroxidase complex. Then sections were developed with chromogen 3,3′ Diaminobenzidine (DAB) at RT and rinsed in tap water for 5 min. This rinsing was then followed by counterstaining with hematoxylin for 5 min and dehydration through several washes of alcohol and xylene. Finally, the slides were cleared and mounted with DPX. Image-Pro Plus 6.0 software (Rockvile, MD, USA) was used to calculate the area percentage of the positive staining [71 (link)].

IHC was also used for localization of biomarkers and to detect differentially expressed proteins when autofluorescence and light scattering interfered with image quality. IHC was used for DLX5, DLX6, HAND2 and P53. For IHC, sections were deparaffinized and rehydrated in a series of ethanol dilutions. The sections were boiled for 30 seconds in 10% blocking solution (Biocare, MX, DV-200), washed with 0.05% PBS-Triton, then incubated with the following primary antibodies: rabbit polyclonal anti-DLX5 (Abcam, MA, YH05211C), polyclonal goat anti-DLX6 (Santa Cruz, CA, sc-18154), monoclonal mouse anti-HAND2 (Santa Cruz, CA, sc-130629), monoclonal mouse anti-P53 (Abcam, MA, GR1277-1), diluted in horse serum overnight at 4 °C. Next day, they were washed with 0.05% PBS-Triton and then incubated with the secondary anti-mouse IgG, anti-rabbit IgG, or anti-goat IgG (Vector lab PK-7800, CA) for 20 minutes at room temperature and then washed with 0.05% PBS-Triton. We then treated the samples with tertiary antibody streptavidin-peroxidase complex (Vector lab SK-4800, CA) for 5 minutes at room temperature. Finally, the sections were treated with chromagen (Impact Novared Kit SK-4805) followed with hematoxylin (Fisher scientific, 123022) for nuclear staining. The hematoxylin was rinsed off with distilled water and the slides mounted with cytoseal (Fisher Scientific, PA).