Signal enhancer hikari

Xenograft tissues were fixed with 4% paraformaldehyde for 4 h and then embedded in paraffin. Sections were then cut to 3–4 μm thickness. For hematoxylin-eosin (HE) staining, deparaffinized sections were stained with Carazzi’s hematoxylin for 10 min, followed by staining with eosin Y for 10 min. For immunofluorescent staining, deparaffinized sections were boiled in ImmunoSaver (Fujifilm Wako) at 98°C for 60 min for antigen retrieval. After blocking with Blocking One Histo (Nacalai Tesque, Inc.) for 60 min, sections were incubated with primary antibodies, including anti-human vimentin (Invitrogen, Carlsbad, CA, USA; cat. no. MA5-11883; 1:50 dilution) and anti-phospho-CREB (Cell Signaling Technology; 1:200 dilution), diluted in Signal Enhancer HIKARI (Immunostain Solution A; Nacalai Tesque, Inc.) overnight at 4°C. After washing, sections were incubated with secondary antibodies (AlexaFluor 568-conjugated goat anti-rabbit IgG [Thermo Fisher Scientific; 1:400 dilution] and AlexaFluor 488-conjugated goat anti-mouse IgG; 1:400 dilution]) and DAPI (5 μg/mL) diluted in Signal Enhancer HIKARI (Immunostain Solution B; Nacalai Tesque, Inc.) for 60 min at room temperature.

Dorsal skin was excised and fixed with 20% formalin solution (Fujifilm Wako Pure Chemical Corporation). The skin samples were embedded in paraffin by a conventional method and stained with hematoxylin and eosin. Images were acquired using an Olympus virtual slide system VS110 (Olympus, Tokyo, Japan). The thickness of the epidermis was expressed as the mean values from five fields per mouse.
For immunohistochemistry, cryosections of mouse dorsal skin were fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corporation) for 10 min, washed with 0.1% Triton X-100 in phosphate-buffered saline for 10 min, and incubated with Blocking One (Nacalai Tesque, Kyoto, Japan) for 30 min. Following overnight incubation at 4 °C with primary antibodies, the sections were incubated with Alexa Fluor-conjugated secondary antibodies at room temperature for 60 min in the dark and mounted in PLUS Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA). Antibodies were diluted with Signal Enhancer HIKARI (Nacalai Tesque). Images were acquired using an LSM 700 confocal laser microscope (Carl Zeiss Co., Ltd., Oberkochen, Germany).

For whole-cell lysates, 3 OD₆₀₀ cells were harvested and resuspended in 15% TCA overnight. About 500-μl 0.5-mm glass beads were added, and samples were vortexed for a total time of 15min, with intermittent cooling on ice. They were centrifuged at 15,000g for 10 min, and the obtained pellet was washed twice with 100% acetone, air-dried, and resuspended in 1× sample loading buffer and boiled for 10 min. Samples were separated on a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P (Merck).
For Supplementary Figs. 1c, 2d, e, and 6g, primary antibody and secondary antibody dilutions were made in skim milk. Proteins bound by antibodies were detected with Clarity western ECL (BioRad) and visualized with Versadoc (BioRad). For Fig. 7g and Supplementary Fig. 1a, 2b, 6a, b, d primary and secondary antibody dilution were prepared in Signal Enhancer Hikari (Nacalai tesque). ChemiDoc Touch (BioRad) was used to visualize proteins reacting with antibody in the presence of the substrate ECL Prime (GE Healthcare). ImageJ^{78 ,79} and Image lab v6.0.0 (BioRad) was used to visualize and process images. Antibodies used are tabulated in Supplementary Table 5. Uncropped blots are presented in the Source Data File.