Ab32363

Immunofluorescence staining was carried out according to the protocol as previously described.23 Briefly, control‐VPCs and MFS‐VPCs were fixed with formaldehyde for half an hour. Following permeation with 0.1% Triton X‐100 in PBS for 30 minutes, cells were stained with ki‐67 antibody (Abcam, ab15580), c‐kit antibody (Abcam, ab32363), γH2AX antibody (Abcam, ab81299) and incubated overnight at 4°C with a 1:100 dilution. After washing with PBS three times, cells were incubated with the secondary antibodies. Finally, the sample was mounted with DAPI and photographed. Images of five different view fields for each slide were captured randomly by a motorized inverted microscope and analysed using AxioVision (Zeiss). The percentage of positive cells was calculated in three independent experiments.

Adenomyosis eutopic endometrium and corresponding ectopic endometrium were collected during surgery. The diagnosis of adenomyosis was confirmed by imaging or histological examination. Samples were collected in the proliferative phase of the menstrual cycle. Sections were incubated with anti-c-kit antibody (dilution 1:200, ab32363, Abcam, Cambridge, MA, USA) and anti-PGP9.5 antibody (dilution 1:500, Z5116, Dako Cytomation, DenmarkA/S). Immunohistochemical assay was performed as previously described 24 (link). Individual nerve fibers were then counted under high power (× 200) to obtain a nerve count in a defined area. The average nerve count in five hot spots was calculated 25 (link).

Paraffin sections from the 200 cases of GC patients were dewaxed in xylene and ethanol. After cleaned in distilled water, the paraffin sections were pretreated with citrate buffer, pH 6.0 (CD177) and EDTA Antigen Retrieval Solution, pH 8.0 (CD4, CD8, CD20, CD56, CD68, and CD117) for 3 min at 120°C in a pressure cooker, and endogenous peroxidase was inhibited with 3% H₂O₂ in PBS for 10 min. Nonspecific actions in the sections were also blocked with goat serum (BOSTER, USA) for 1 h at room temperature. The sections were then incubated with the primary antibody overnight at 4°C, followed by incubation with the secondary antibody for 30 min at 37°C. Primary antibodies used were CD4 (ab183685, 1 : 1000, Abcam, Cambridge, MA, USA), CD8 (ab4055, 1 : 100, Abcam, Cambridge, MA, USA), CD20 (ab9475, 1 : 50, Abcam, Cambridge, MA, USA), CD56 (ab75813, 1 : 100, Abcam, Cambridge, MA, USA), CD68 (ab213363, 1 : 4000, Abcam, Cambridge, MA, USA), CD117 (ab32363, 1 : 400, Abcam, Cambridge, MA, USA), and CD177 (ab220279, 1 : 200, Abcam, Cambridge, MA, USA). Second antibodies used were goat anti-rabbit IgG (CD4, CD8, CD56, CD68, CD117, and CD177) and goat anti-mouse IgG (CD20). The chromogenic reaction was performed via diaminobenzidine (DAB) staining, and the staining intensity was measured using Image-Pro Plus version 6.2 software (Media Cybernetics, Rockville, Maryland, USA).