Simple stain rat max po

Paraffin-embedded sections (4-μm thickness) from testis and epididymis samples fixed in 4% PFA/PBS were subjected to hematoxylin and eosin or immunohistochemical staining. For
immunohistochemistry, deparaffinized tissue sections were treated with 10% hydrogen peroxide in methanol at room temperature for 5 min to quench endogenous peroxidase activity. Antigen
retrieval was achieved by autoclaving sections in citrate buffer (pH 6.0) for 20 min. Sections were blocked with 5% normal goat serum (NGS) in PBS for 20 min at room temperature.
Subsequently, sections were incubated overnight at 4°C with either anti-vimentin (clone V9, undiluted, ready-to-use; DAKO-Agilent Technology, Santa Clara, CA, USA) or anti-DTSP (diluted
1:2,000 in 5% NGS/PBS) antibodies. Primary antibodies were detected using an HRP-conjugated secondary antibody (Simple stain rat MAX-PO, undiluted, ready-to-use; Nichirei, Tokyo, Japan).
After incubation with an HRP-conjugated secondary antibody, 3-3ʹ-diaminobenzidine served as the chromogen. Nuclei were counterstained with hematoxylin. Immunostained sections were observed
under a microscope equipped with a digital camera (DP73).

Procedures for immunohistochemistry, including antibodies used as primary antibodies, are
summarized in Table 1Table 1.. Formalin-fixed and paraffin-embedded tissues were deparaffinized and
rehydrated through graded alcohols. Endogenous peroxidase activity was quenched with 3%
H₂O₂, followed by heat-induced antigen retrieval in a microwave at
95°C with citrate buffer at pH 6.0. After blocking endogenous peroxidase activity,
sections were treated with serum-free protein block (Dako Japan, Tokyo, Japan) for 30 min
at room temperature. Sections were then washed with washing buffer (Dako Japan, Tokyo,
Japan) and reacted with the primary antibody overnight at 4°C. Sections were washed and
treated by applying drops of Simple Stain Rat MAX PO (MUITI) (Nichirei Biosciences, Tokyo,
Japan) to sections reacted with S-100, αSMA, CD10, Iba-1, and proliferating cell nuclear
antigen (PCNA) primary antibodies for 10 min at room temperature or by applying drops of
Simple Stain Mouse MAX-PO (G) (Nichirei Biosciences) to sections reacted with desmin for
30 min at room temperature. After washing sections with washing buffer, reactions were
visualized using 3,3′-diaminobenzidine (DAB) as a chromogen. Sections were lightly
counterstained with hematoxylin.