Hematoxylin and eosin

Primary sebocyte cultures were obtained from occipital hair follicle sebaceous glands. From the hair follicles, the sebaceous glands were dissected under a binocular microscope and transferred to a tissue culture dish. The cells were maintained in Dulbecco's modified Eagle medium (DMEM; Hyclone Laboratories, Logan, UT, USA) at 37℃ in a humidified atmosphere of 5% CO₂. Explants were incubated for three days, and the medium was then changed to Epilife (MEPI500CA; Gibco BRL, Grand Island, NY, USA). The medium was changed every three days. DMEM was supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and 20% heat inactivated bovine serum (Hyclone Laboratories), while Epilife was supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), and fungizone (250 µg/ml).
Once the cells became subconfluent, they were harvested using 0.25% trypsin and 10 mM ethylenediamine tetraacetic acid (EDTA) in Hank's balanced salt solution, followed by subculturing at a split ratio of 1:3. Cells obtained after the second passage were used in this study. These cultured sebocytes were subjected to hematoxylin and eosin (Muto Pure Chemicals Co., Tokyo, Japan) and Oil Red O (Sigma, St. Louis, MO, USA) staining, and immunocytofluorescence against cytokeratin 1 and 7 (Chemicon, Billerica, MA, USA) before experimental use (Fig. 1).

Thymus tissues were fixed with 4% (g/vol) paraformaldehyde (PFA) and embedded in OCT optimum cutting temperature compound (Sakura Finetek). Frozen thymuses were sliced into 10-μm-thick sections and stained with antibodies specific for β5t (Murata et al., 2007 (link); MBL), Aire (Invitrogen, clone 5H12), and LC3B (Cell Signaling Technology, clone D11), followed by AlexaFluor-conjugated anti-IgG antibodies (Invitrogen). Sections were also stained for the reactivity with UEA1 (Vector Laboratories). For the staining with antibodies specific for CD4 (Invitrogen, clone RM4–5) and CD8α (Invitrogen, clone 53–6.7) in addition to the staining for the reactivity with UEA1, thymus tissues were embedded, sliced, and fixed with acetone. Images were visualized and analyzed with a TCS SP8 (Leica) or an ECLIPSE Ti2 (Nikon) confocal laser scanning microscope.
For the measurement of the area of thymic regions, frozen thymuses were sliced into 10-μm-thick sections, fixed with neutral buffered formalin, stained with hematoxylin and eosin (Muto Pure Chemicals), and imaged under an Eclipse E1000 microscope (Nikon). The areas of the cortical and medullary regions in the thymic sections were measured by using Photoshop software (Adobe).