Alp stain kit

BMSCs cultured in different groups for 14 days were collected and lysed. After centrifugation, ALP activity was assayed in the BMSC lysates using a commercial ALP assay kit, (P0321, Beyotime), and the results were read on a microplate reader (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA) [18 (link)]. BMSCs were also stained with an ALP Stain Kit (G1480, Kaplow’s/Azo Coupling Method, Solarbio, Beijing, China) and observed under a microscope.

Human adipose-derived MSCs were cultured in minimum essential medium α (Gibco, 12571063) supplemented with 10% bovine growth serum (Gibco, 10099141C) and 1% penicillin/streptomycin (Gibco, 15140122) at 37 °C with 5% CO₂. Glass coverslips without hydrogels were also incubated in collagen I (ThermoFisher, A1048301) for 10 min before seeding cells. For inhibitor experiments, drugs and corresponding concentrations are as follows: blebbistatin (50 μM, Sigma, B0560), nocodazole (0.1 μM, Abcam, ab1230630), and latrunculin B (1 μM, Abcam, ab144291). Inhibitors remained in the media during cell culture. Transfections were carried out with Lipofectamine RNAiMax (Invitrogen) for small interfering RNA. The sequences of small interfering RNA used in this study were shown in Table S1. CCK8, caspase-3 assay kit (Colorimetric) (Abcam, ab39401), and ALP stain kit (Solarbio, G1480) were used according to the protocol.

After baking and dewaxing, paraffin sections were dipped in double distilled water. Then, sections were stained with an ALP Stain Kit (Solarbio, Beijing, China) or a PAS Kit (Maixin, Fuzhou, China). Nuclear fast red was used to counterstain the nuclei in ALP staining, and haematoxylin was used to counterstain the nuclei in PAS staining. ALP staining was used to visualize the apical brush border of epithelial cells [22 (link)]. The goblet cells were labelled with PAS staining, and the number of PAS⁺ goblet cells in each section was counted by two experimenters blinded to the experimental design. At least 15 villi and crypts were counted randomly for each slide and the mean value was calculated.