Rabbit anti slug

Triton X‐100 lysis buffer [20 mm Tris (pH 7.4), 2 mm EDTA, 150 mm sodium chloride, 1 mm sodium deoxycholate, 1% Triton X‐100, 10% glycerol, 2 pills protease inhibitor cocktail (Roche)] was used to collect protein from cells. Protein samples were heat‐denatured and equally loaded, separated on 8–12% SDS/PAGE gel, transferred onto a polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK), and blocked with 5% nonfat dry milk. Primary antibodies for western blot analyses included mouse anti‐α‐tubulin (1 :  20 000 dilution; Sigma), rabbit anti‐Snail (1 : 1000 dilution; Cell Signaling, Danvers, MA, USA), rabbit anti‐Slug (1 : 1000 dilution; Cell Signaling), rabbit anti‐CHIP (1 : 1000 dilution; Abgent, San Diego, CA, USA), mouse anti‐Flag (1 : 3000 dilution; Abcam), mouse anti‐GFP (1 : 1000 dilution; Santa Cruz), mouse anti‐HA (1 : 1000 dilution; Abcam, Cambridge, MA, USA), mouse anti‐Vimentin (1 : 1000 dilution; Santa Cruz, Santa Cruz, CA, USA), mouse anti‐E‐cadherin (1 : 5000 dilution; BD Biosciences, San Jose, CA, USA), and mouse anti‐Ub (1 : 5000 dilution; Santa Cruz). Membranes were incubated with horseradish peroxidase‐conjugated anti‐mouse or anti‐rabbit secondary antibody (1 : 5000 dilution; Cell Signaling) for 1 h, and chemiluminescence signals were detected by ECL substrate (GE Healthcare, Chicago, IL, USA).

The protein were separated and extracted from eutopic endometrium in control group and ectopic endometrium in other groups. The tissue lysates were prepared as described previously (Chen et al., 2015 (link), 2017 (link)). Briefly quantified protein lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore, United States) and probed with primary rabbit anti-MMP-2, rabbit anti-MMP-9 (1:100 dilution; Boster Biological Technology, Wuhan, China), rabbit anti-TIMP-1 (1:300 dilution; Proteintech Biotechnology, Wuhan, China), rabbit anti-E-cadherin, rabbit anti-Vimentin, rabbit anti-Snail, rabbit anti-Slug (1:1000 dilution; Cell Signaling Technology, Beverly, MA, United States), rabbit anti-β-actin (1:5000 dilution; Proteintech Biotechnology, Wuhan, China) overnight at 4°C. Then the membranes were blotted with an appropriate horseradish peroxidase-linked goat secondary antibody (1:2000 dilution; Zhongshan Golden Bridge Biotechnology, Beijing, China). Electrochemiluminescence was performed according to the manufacturer’s instructions with Tanon 5200 imaging system (Tanon, China). β-Actin was used as endogenous control.

Cultivated cells were fixed for 20 min using 4% paraformaldehyde (PFA), washed and permeabilized in PBS with 0.02% TritonX-100 (Sigma Aldrich) and supplemented with 5% goat serum for 30 min. The applied primary antibodies were diluted in PBS as followed: rabbit anti-Nestin 1:200 (Millipore), mouse anti-S100B 1:500 (Sigma Aldrich), rabbit anti-Slug 1:100 (Cell-Signaling Technology), rabbit anti-p75 1:500 (Cell-Signaling Technology), mouse anti-β-III-tubulin 1:100 (Promega), rabbit anti-neurofilament-L 1:50 (Cell-Signaling Technology), anti-vGlut (Millipore) and anti-Synaptophysin (Millipore). They were applied for 1 h (cells) at room temperature. After three washing steps, secondary fluorochrome-conjugated antibodies (Alexa 555 anti-mouse or Alexa 488 anti-rabbit, Invitrogen, Life Technologies GmbH) were applied for 1 h at RT with a dilution ratio of 1:300. Nuclear staining was realized by incubation with 4,6-Diamidin-2-phenylindol (DAPI) (1 μg/mL, Applichem) in PBS for 15 min at RT. Finally, the samples were mounted with Mowiol (self-made). Imaging was performed using a confocal laser scanning microscope (CLSM 780, Carl Zeiss) and image processing was executed with ImageJ and CorelDRAW [48 (link)] (open source and Corel Corporation).