Alkaline phosphatase

IHC staining was performed on 5 µm FFPE sections, extracted from the same frozen sections from which DNA and RNA were extracted. Slides were deparaffinised in series of xylene and hydrated in a series of descending ethanol. Heat-induced antigen retrieval was performed using TRIS-EDTA (pH = 9), followed by immunostaining performed on the Leica Bond III autostainer (Leica Biosystems). Antibodies used included mouse monoclonal anti-human CD3 (DAKO, clone F7.2.38, dilution 1:50) and mouse monoclonal anti-human CD8 (DAKO, clone C8/144B, dilution 1:25) at 1 h RT. DAKO REAL^TME alkaline phosphatase and chromogen red detection system was used for secondary detection of positive staining. Stained slides were counter-stained with haematoxylin and cover-slipped for review. Image acquisition was performed on the Hamamatsu whole slide scanner at 40-fold magnification.

The tissues were fixed with 4% paraformaldehyde-phosphate buffer solution and embedded in paraffin. Sections were taken from the central portion of the wound and stained with hematoxylin-eosin (HE) according to the standard method.
For immunohistochemical analysis, after endogenous peroxidase was blocked with methanol/hydrogen peroxide, the sections were incubated with 10% normal rabbit serum for 20 min to block non-specific binding and then stained with anti-α-smooth muscle actin (α-SMA) antibody (dilution 1:200; Vector Laboratories, Inc., Burlingame, CA, USA), anti-Ly6G Ab (clone 1A8; dilution 1:100; BioLegend), or anti-MMP-2 (dilution 1:200; Chemicon, Darmstadt, Germany). The sections were incubated with peroxidase-conjugated secondary Ab (4 µg/mL; Histofine Simple Stain MAX-PO, Nichirei Bioscience, Tokyo, Japan), then reacted with 3, 3-diaminobenzidine (DAB) (Nichirei Bioscience) or Alkaline Phosphatase (Dako, Bettingen, Switzerland). The number of myofibroblasts and neutrophils in six random fields (each 0.2 mm²) was determined by counting the number of α-SMA-positive cells or the number of Ly6G-positive cells, respectively. All analyses were performed under blinded conditions.

Whole testes from mice were fixed in Bouin’s fluid for 8 hours. Tissues were dehydrated in a graded series of ethanol and embedded in paraffin. Sections that were 7 μm thick were processed for stainings. Sections were deparaffinized and rehydrated before antigen retrieval, followed by processing for antibody staining against the Leydig cell marker protein INSL3 (orb18041, AA range: 10–50, 1:100, Biorbyt) or the macrophage marker protein F4/80 (MCA497GA, Cl:A3-1, 1:500, AbD). The stainings were developed using alkaline phosphatase (Dako, 1:200) followed by counterstain with Harris’ hematoxylin and mounting under VectaMount AQ (Vector Laboratories). For immunofluorescence on tissues, sections were immunostained with antibodies against STAR (8849, 1:1000, clone D10H12, Cell Signaling Technology) or CCL2 (orb36895, 1:1000, Biorbyt) antibodies followed by detection with secondary antibodies (anti–rabbit IgG, 711295152, 1:400, Jackson ImmunoResearch; or anti–mouse IgG, SAB3701033, 1:400, MilliporeSigma) and counterstained with DAPI. For immunofluorescence on cells, cell smears were fixed in 4% paraformaldehyde and permeabilized in 70% ethanol. After that, slides were immunostained with antibodies against STAR, CCL2, or VDAC-1 (SAB5201374, clone S152B-23, 1:200, MilliporeSigma) followed by conjugation with the abovementioned secondary antibodies and counterstain with DAPI.