Ab150077 ab150113

The paraffin‐embedded intact uterine samples in which the conceptuses were not flushed out were cross‐sectioned into 4 μm thick using a Leica microtome (RM2235, Leica, Germany) and then deparaffinized and rehydrated by passage through xylene and a grade alcohol. Sections that were stained with haematoxylin and eosin were used for histological analysis.
Immunofluorescence assays were carried out as described by Wang et al.⁶ The primary antibodies used were ST6GAL1 (1:50, ab77676, abcam), ST6GAL2 (1:50, AF7747; R&D Systems), E‐cadherin (1:50, ab40772; abcam), β‐catenin (1:50, ab6302; abcam), Vimentin (1:50; sc‐73258), Rac1 (1:30, 66122–1‐Ig; Proteintech) and RhoA (1:50, 10749–1‐Ig; Proteintech). The fluorescent‐dye conjugated secondary antibodies used were Alexa Fluor^® 488 (1:100, ab150077/ab150113; abcam) or Alexa Fluor^® 555 (1:100, A21422/A21428; invitrogen). All slides were scanned by a Pannoramic Midi slide scanner (3D HISTECH; Budapest), and the image analysis was carried out by using CaseViewer 2.0 software (3D HISTECH).

Tissues were first washed with PBS buffer and adventitia was removed carefully, and then they were fixed with 4% paraformaldehyde (PFA) and embedded in 20% sucrose solution before being frozen in TissueTek cutting medium (Sakura Finetek). 8 μm sections were processed for immunofluorescent analysis. The sections were further fixed with 4% PFA for 20 minutes. For immunostaining of attached cells, cells were fixed in 4% PFA for 20 minutes and permeabilized with 0.1% Triton X-100 (in PBS) for 5 minutes and rinsed for 3 times. Nonspecific binding was blocked by 4% BSA in PBS for 1 hour. Tissues/cells were incubated at 4 °C overnight in incubation buffer containing 4% BSA and the primary antibodies including anti-CDH5 (1:100), anti-Ki67 (Abcam, ab15580, 1:200), anti-F4/80 (BioLegend, 157309, 1:100), anti-SM22α (Proteintech, 10493-1-AP, 1:100), anti-ALDH2 (1:100), anti-ALDH3A1(1:100), anti-ALDH6A1 (1:100), and anti-pSmad2/3 (1:100). After being washed in PBS for 3 times, the specimens were incubated with Alexa Fluor 488-conjugated goat anti-rabbit/-mouse IgG (Abcam, ab150077/ab150113) or Alexa Fluor 555-conjugated goat anti-rabbit/-mouse IgG (Abcam, ab150078/ab150114) or Alexa Fluor® 647 goat anti-rabbit igG (Abcam, ab150083) for 1 hour at room temperature. The fluorescent signals were detected by fluorescence microscopy (Leica TCS SP8).

For immunofluorescence, cells were fixed in 4% PFA for 15 min and permeabilized with 0.2% Triton X-100 in PBS for 5 to 10 min. Nonspecific binding was blocked by 3% BSA for 30 min. The primary antibody was diluted 1:200 in 3% BSA and incubated overnight at 4 °C, then probed with secondary antibody including Alexa Fluor 488-conjugated goat anti-rabbit/-mouse IgG (ab150077/ab150113; Abcam) or Alexa Fluor 555-conjugated goat anti-rabbit/-mouse IgG (ab150078/ab150114; Abcam) diluted 1:200 in PBS for 1 h at room temperature. Nuclei were counterstained with DAPI. Images in either fixed cells or live cells were captured by using Leica SP8 confocal microscope or stimulated emission depletion (STED) super-resolution microscopy.
For quantification of subcellular distribution, the regions of cell nucleus or cytoplasm were manually determined, and measurements of fluorescence intensity were conducted by using NIH ImageJ software. Statistic data were obtained from approximate 100 ~ 200 cells from 18 random fields from 3 independent experiments. Each dot represents the mean value of all the cells from one random field.