Dp72 digital imaging system

After deparaffinization, tissue sections were incubated in a 0.01 m citrate buffer (pH 6.0) at 121°C for 15 min to retrieve the antigenicity of the relevant proteins. The sections were then immersed in 0.3% H₂O₂ and 0.1% sodium azide (Wako Pure Chemical Industries) in phosphate‐buffered saline (1/15 m, pH 7.4) for 25 min to block endogenous peroxidase activity. After incubation in 10% normal goat serum (Vector Laboratories), sections were treated with antibodies against podoplanin (1:100, D2‐40; Dako) and CD31 (1:250, EP3095; Abcam) at room temperature overnight. After washing in phosphate‐buffered saline, sections were incubated with alkaline‐phosphatase‐conjugated anti‐mouse IgG (Histofine Simple Stain AP, Mouse; Nichirei Bioscience) for 1 h at room temperature as a secondary antibody for podoplanin immunostaining. The blue immunoreaction was visualized using an alkaline phosphatase reaction (Vector blue substrate kit, Vector Laboratories). Sections were then incubated with peroxidase‐conjugated anti‐rabbit IgG (Histofine Simple Stain MAX‐PO, Rabbit; Nichirei Bioscience) for 1 h at room temperature. The brown immunoreaction for CD31 was visualized using 3,3'‐diaminobenzidine reaction (Wako Pure Chemical Industries). The stained sections were examined using a BX‐60 light microscope equipped with a DP72 digital imaging system (Olympus).

The liver and hindgut tissues fixed in paraformaldehyde were removed and washed, and then sections were made according to routine procedures. Liver tissue sections were stained with H&E, Masson, and oil red O, and intestinal tissue sections were stained with H&E. An Olympus BX51 microscope and DP72 digital imaging system were used for observation.

Skin tissues were prepared for histological sectioning following Carter and Clarke^{46 (link)}. Briefly, skin tissues were fixed in a 4% paraformaldehyde solution and embedded in paraffin wax. The paraffin-embedded tissue blocks were sectioned into 5-μm-thick slices using a Leica RM2255 Automated Rotary Microtome (Germany), which were then stained with HE staining. Morphological observations were conducted with an Olympus BX51 microscope (Olympus, Japan), and digital images were acquired using an Olympus DP72 digital imaging system (Japan). Upon observation at 40x magnification, follicles with a red-stained IRS were defined as active SFs. Five fields of each sample were observed. Based on these observations, the total number of active SFs was counted in each group (N = 4).