Tris buffered saline (tbs)

The grafted skin tissues were embedded in Tissue-Tek® Optimal Cutting Temperature compound (Sakura Fineteck, Tokyo, Japan) and sectioned at 10-μm thickness. The sections were washed thrice with Tris-buffered saline (TBS, TaKaRa Bio, Shiga, Japan) for 10 min and blocked for 1 h in TBS (TaKaRa Bio) containing 5% donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and 0.3% Triton X-100 (FUJIFILM Holdings Corporation, Tokyo, Japan). The sections were then probed overnight at 4 °C with anti-CD3 (CD3-12; Abcam, Cambridge, UK, USA) and anti-CD45 (L12/201; Bio-Rad) antibodies in TBS containing 1% donkey serum and 0.3% Triton X-100. Subsequently, samples were again washed thrice with TBS for 5 min and were labelled for 1 hour at room temperature (20 °C–28 °C) with secondary antibodies conjugated with Alexa Flour 488 (Thermo Fisher Scientific). The sections were counterstained with Hoechst 33342 (Thermo Fisher Scientific) to visualise the nuclei and imaged using a fluorescence microscope (Axio Observer. D1, Carl Zeiss, Jena, Germany).

Total cellular proteins were prepared as previously described [19 (link)]. For western blot analysis, 20 mg of cellular protein was subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) on a 4–15% gradient gel (Bio-Rad). The blot was transferred onto a polyvinylidene difluoride membrane using the iBlot 2 (Life Technologies), followed by blocking with Tris-buffered saline (Takara Bio, Shiga, Japan) containing 0.2% dry fat-free milk (Cell Signaling). Primary antibody reaction, horseradish-peroxidase-conjugated secondary antibody (NA934V, GE Healthcare UK Ltd., Buckinghamshire, UK) reaction, and washing steps have been previously described [30 (link)]. Bands were visualized using Amersham ECL western blotting detection reagent (GE Healthcare UK Ltd) and the ChemiDoc XRS Plus ImageLab system (Bio-Rad).

Total cellular protein was prepared as described previously [29 (link)], and the protein concentration was measured using Quick Start Bradford Reagent (Bio-Rad, Hercules, CA, USA). Twenty micrograms of protein was subjected to sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) in a 4 to 20% gradient gel (Bio-Rad), and the blot was transferred onto a polyvinylidene difluoride membrane by iBlot 2 (Life Technologies, Carlsbad, CA, USA). After blocking with 0.2% nonfat dry milk (Cell Signaling Technology, Danvers, MA, USA) in Tris-buffered saline (Takara Bio), the membrane was incubated with the primary antibodies (anti-TPD52 antibody (1/1,000 dilution; Abcam, Branford, CT, USA), anti-TPD54 antibody (1/1,000 dilution), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1/10,000 dilution; Sigma-Aldrich, St. Louis, MO, USA) and the horseradish-peroxidase-conjugated secondary antibody (GE Healthcare UK Ltd., Buckinghamshire, UK)) as described previously [29 (link)]. The protein bands were visualized using Amersham ECL Western Blotting Detection Reagents (GE Healthcare) and a Chemidoc XRS Plus ImageLab System (Bio-Rad).