Pentobarbital sodium salt

Enzyme-linked immunosorbent assay (ELISA) plates were purchased from AGC (Tokyo, Japan). Horseradish-peroxidase (HRP)-conjugated goat antimouse IgE antibody was purchased from Southern Biotech (Birmingham, AL, USA). HRP-conjugated goat antimouse IgG1-antibody was purchased from Bethyl Laboratories (Montgomery, TX, USA). 3,3′,5,5′-tetramethylbenzidine (TMB) peroxidase substrate was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD, USA). Coomassie Brilliant Blue R-250, Bradford protein assay reagent, and pentobarbital sodium salt were purchased from Nacalai Tesque (Kyoto, Japan). Enhanced chemiluminescence (ECL) Western blotting substrate and X-ray films (Amersham Hyperfilm MP) were purchased from GE Healthcare (Chalfont St. Giles, UK). Immunoreactive enhancers, CanGet Signal solutions 1 and 2, were purchased from TOYOBO (Osaka, Japan). All other chemicals used in this study were of the highest purity available.

Mice were treated in accordance with the Guidelines for the Proper Conduct of Animal Experiments (Science Council of Japan), and the Institutional Animal Care and Use Committee approved the study. Male C3H/He inbred mice at 7 weeks of age (Japan SLC Inc., Shizuoka, Japan) were acclimatized at 3–5 mice per cage for 2 weeks in an animal room maintained at 23°C and 55% humidity. Mice were housed on a 12 h:12 h light:dark schedule, with white light on at 10–50 lux during the day. In the nighttime handling of mice, procedures were performed under blue LED light of <0.4 lux. The radioprotector cysteamine HCl (Wako Pure Chem. Co., 3 mmole/kg) and the anesthetic pentobarbital sodium salt (Nacalai Tesque Co., 76 mg/kg) were injected subcutaneously.

After 1, 2, and 4 weeks, the rats were euthanized using an intraperitoneal overdose of anesthetic (Pentobarbital sodium salt, Nacalai tesque, Inc., Kyoto, Japan). Skin tissue, including wounds, was collected from rats for histological evaluation. The excised tissues were fixed with 20% paraformaldehyde-containing phosphate buffer for 48 h. The fixed tissues were then embedded in a paraffin wax block. The specimens were sectioned into 6–8 µm thicknesses from the block. The sections were then stained using hematoxylin and eosin staining (HE staining: Muto Pure Chemicals, Co., Ltd., Tokyo, Japan) and Masson’s trichrome staining (MT staining: Muto Pure Chemicals, Co., Ltd., Tokyo, Japan). Cells and collagen generated during wound healing were observed on the stained sections with an optical microscope (BX53, OLYMPUS, Tokyo, Japan).