Ba210t

Hematoxylin (AWI0001a, Abiowell, China) was stained for 1 ∼ 10 min, rinsed with distilled water and returned to blue in PBS (AWI0129a, Abiowell, China). Eosin (AWI0029a, Abiowell, China) was stained for 1 ∼ 5 min and rinsed with distilled water. Gradient alcohol (95–100%) was used for dehydration for 5 min per stage. Neutral gum (AWI0238a, Abiowell, China) was sealed and observed by microscopy (BA210T, Motic, China).

8 PDP patients and 4 relative normal controls’ paraffin embedded gastric mucosa tissues were performed immunohistochemical staining. Specimens were grilled for 2 hours at 60° C oven at first. Then the specimens were dewaxed with xylene and rehydrated with gradient alcohol. Antigen retrieval was performed by using 0.01 mmol citrate buffer to make the samples infiltrate in for 15 minutes after steaming in pressure-cooker. After retrieval, the tissues were dealt with rabbit antibody against human IL-6 (abcam, ab9324) and mouse antibody against human RANKL (abcam, ab45039) and TNFα (abcam, ab6671) overnight at 4 °C. Biotinylated secondary antibody was adopted in SP kit at room temperature for 10 minutes followed with incubating of an appropriate amount of HRP-conjugated secondary antibody at room temperature for 30 minutes. Immunostaining was carried out through using DAB for chromogen and hematoxylin for nuclear staining. The specimens were cleared in xylene after dehydrated with gradient alcohol. Finally the neutral beams were applied to seal the specimens. All immunostained specimens were examined with the microscope (BA210T, Motic) and assessed by two pathologists blinding to clinical features. Under the Microscopy, the images and IOD (integrated optical density) were acquired with the Image-Pro Plus software 6.0.

Epididymal white adipose tissues (WAT) and livers were fixed with 4% paraformaldehyde for 24 hr and embedded in paraffin. Then, 5‐μm sections were prepared and stained with hematoxylin and eosin (H&E). The physiology of epididymal WAT and livers were observed by inverted microscope (Motic BA210T, China).

Liver tissues were fixed in 10% neutral formalin for 24–48 h, dehydrated by an ethanol gradient (70–100%), cleared by xylene, embedded in paraffin and cut into slices of 5 μm. Then the slices were stained with hematoxylin and eosin (HE), placed on a glass dish, sealed with a neutral gum, and examined under a light microscope (BA210T, Motic, China) at a magnification of 400×.^{31 (link)}

The abdominal aortic tissue samples of mice were taken and fixed in 4% paraformaldehyde for 24 h, followed by gradient dehydration with 20% and 30% sucrose solutions. The abdominal aortic tissue was sliced, dehydrated, embedded in paraffin, successively sliced with a paraffin slicer, connected to the treated glass slides, and baked at 60°C for 12 h. The slices were placed in xylene for 20 min × 3 times. Then, the slices were placed in 100%, 95%, 85%, and 75% ethanol successively for 5 min at each stage for dewaxing to water. Hematoxylin (Wellbio, Changsha, China) was dyed for 3 min, washed with distilled water, and PBS was returned to blue. The slices were dyed with eosin (Wellbio, Changsha, China) for 5 s and rinsed with distilled water. The slices were soaked in gradient alcohol (95–100%) and dehydrated for 5 min per grade. After removal, the slices were placed in xylene for 10 min × 2 times, sealed with neutral gum, and observed under a microscope (BA210T, Motic).

The rat heart tissues were taken to make paraffin sections. The slices were roasted at 60°C for more than 3 h, and dewaxed to water. The appropriate amount of nuclear dye was added to cover the whole tissues, and stained for 3–5 min. We rinsed the staining solution completely with tap water, soaked the slices with distilled water, and then soaked the slices with weak alkaline solvent such as PBS or ammonia for 5–10 min to make the nuclei blue again. We removed the water from the slices, dropped the dye to cover the whole tissues, stained them for 6–8 min, and rinsed the dye with rinse solution. The samples were sealed with neutral gum. The results were observed under a microscope (BA210T, Motic, China).

The different distribution and expression levels of TP63 in RWPE-1 and DU145 cell lines was detected by immunofluorescence. The cells were fixed by 4% paraformaldehyde for 30 minutes and incubated with primary anti-p63 antibody (1:50, Abcam#ab124762, Cambridge, UK) at 4°C for a night and incubated with CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (1:100, Proteintech#SA00013-2, Rosemont, USA) at 37°C for 90 minutes. DAPI dye was used to counterstain the cells for 10 minutes. All cells were observed and photographed by microscope (Motic#BA210T).