immunofluorence staining  (Proteintech)

Journal: Materials Today Bio

Article Title: Piezo1 mediated scaffold-free rapid generation of self-mineralized bone organoids via activating Wnt signaling

doi: 10.1016/j.mtbio.2025.102620

Figure Lengend Snippet: Schematic illustration showing the generation, mechanism, and application of self-mineralized Yoda1+ BOs from Piezo1-activated SHED spheroids .

Article Snippet: After blocking with 5 % milk blocking buffer, the membranes were incubated with the primary antibodies for PIEZO1 (Proteintech, China), WNT7b (Abclonal, China), RUNX2 (Abcam, UK) and GAPDH (Proteintech, China) overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies (Invitrogen, GA, USA) for 1 h at room temperature.

Techniques:

Journal: Materials Today Bio

Article Title: Piezo1 mediated scaffold-free rapid generation of self-mineralized bone organoids via activating Wnt signaling

doi: 10.1016/j.mtbio.2025.102620

Figure Lengend Snippet: Piezo1 activation promotes SHED spheroids assembly through up-regulating adhesion molecules. (A) Illustration of the preparation process of Yoda1+ SEHDs spheroids. (B) Optical microscopy images showing the formation of SHED spheroids within 48 h. (C) The cell viability and morphology of cell spheroids observed by HE staining and live/dead staining. (D) & (E) Immunofluorescence staining to visualize the expression of Piezo1 and the morphology of F-actin cytoskeleton in SHED spheroids. (F) & (G) IHC staining and quantitative analysis to visualize the expression of E-cadherin and N-cadherin. (n ≥ 3) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: After blocking with 5 % milk blocking buffer, the membranes were incubated with the primary antibodies for PIEZO1 (Proteintech, China), WNT7b (Abclonal, China), RUNX2 (Abcam, UK) and GAPDH (Proteintech, China) overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies (Invitrogen, GA, USA) for 1 h at room temperature.

Techniques: Activation Assay, Microscopy, Staining, Immunofluorescence, Expressing, Immunohistochemistry

Journal: Materials Today Bio

Article Title: Piezo1 mediated scaffold-free rapid generation of self-mineralized bone organoids via activating Wnt signaling

doi: 10.1016/j.mtbio.2025.102620

Figure Lengend Snippet: Piezo1 activation enhances the osteogenic potential of SHED spheroids . (A) hBMSCs were cultured with conditioned media of Yoda1+ SHED spheroids. (B) The expressions of RUNX2 and BMP2 genes related to osteogenic differentiation. (C) & (D) IF staining and quantitative analysis to visualize the expression of RUNX2 and BMP2. (E) Wound healing assay and corresponding quantitative analysis on cell migration of hBMSCs. (F) ALP staining and Alizarin red staining. (n ≥ 3) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: After blocking with 5 % milk blocking buffer, the membranes were incubated with the primary antibodies for PIEZO1 (Proteintech, China), WNT7b (Abclonal, China), RUNX2 (Abcam, UK) and GAPDH (Proteintech, China) overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies (Invitrogen, GA, USA) for 1 h at room temperature.

Techniques: Activation Assay, Cell Culture, Staining, Expressing, Wound Healing Assay, Migration

Journal: Materials Today Bio

Article Title: Piezo1 mediated scaffold-free rapid generation of self-mineralized bone organoids via activating Wnt signaling

doi: 10.1016/j.mtbio.2025.102620

Figure Lengend Snippet: Piezo1 activation enhances the angiogenic potential of SHED spheroids . HUVECs were cultured with conditioned media of Yoda1+ SHED spheroids. (A) IHC staining and quantitative analysis to visualize the expression of CD31. (B) The expressions of ANG1 and CD31 genes related to osteogenic differentiation. (C) Wound healing assay and corresponding quantitative analysis on cell migration of HUVECs. (D) In vitro tube formation of HUVECs for neovascularization with corresponding quantitative analysis. (n ≥ 3) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: After blocking with 5 % milk blocking buffer, the membranes were incubated with the primary antibodies for PIEZO1 (Proteintech, China), WNT7b (Abclonal, China), RUNX2 (Abcam, UK) and GAPDH (Proteintech, China) overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies (Invitrogen, GA, USA) for 1 h at room temperature.

Techniques: Activation Assay, Cell Culture, Immunohistochemistry, Expressing, Wound Healing Assay, Migration, In Vitro

Journal: Materials Today Bio

Article Title: Piezo1 mediated scaffold-free rapid generation of self-mineralized bone organoids via activating Wnt signaling

doi: 10.1016/j.mtbio.2025.102620

Figure Lengend Snippet: RNA sequencing of Yoda1+ SHED spheroids. (A) Volcano plot of significantly upregulated and downregulated genes (Yoda1+ vs. Yoda1-). (B) The clustered heatmap of differentially expressed genes in Yoda1+ SHED spheroids compared with Yoda1- SHED spheroids related to mechanical stimulus. (C) KEGG enrichment analysis. (D) The clustered heatmap of differentially expressed genes in Yoda1+ SHED spheroids compared with Yoda1- SHED spheroids related to Wnt signaling pathway. (E) Western blot and quantitative analysis of the expression of markers including Piezo1, WNT7B and RUNX2. (F) The schematic illustration that under Yoda1 stimulation, the Piezo1 channel is activated, triggering the activation Wnt signaling pathway and increased expression of RUNX2. (n ≥ 3) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: After blocking with 5 % milk blocking buffer, the membranes were incubated with the primary antibodies for PIEZO1 (Proteintech, China), WNT7b (Abclonal, China), RUNX2 (Abcam, UK) and GAPDH (Proteintech, China) overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies (Invitrogen, GA, USA) for 1 h at room temperature.

Techniques: RNA Sequencing, Western Blot, Expressing, Activation Assay