Cells (3 × 104/cm2) were cultured in a 12-well plate, and RUNX2, COL1A1, and p-AKT were detected using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany) on day 3 after the induction of osteogenesis. Briefly, BMSCs were fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature. Then cells were permeabilized for 30 min in 0.05% Triton X-100 and blocked with 5% bovine serum albumin (BSA) for another 30 min. Fixed cells were then washed 3 times with PBS and incubated at 4 °C overnight with anti-RUNX2 (1:1600; CST), COL1A1 (1:500; Abcam, Shanghai, China), or p-AKT (1:400; CST). Cells were then incubated with a fluorescence-conjugated secondary antibody (Beyotime) for 2 h at room temperature, and the nuclei were then stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 4 min. The results of IF were then observed and recorded under a fluorescence microscope (Leica, Solms, Germany).
Col1a1
Manufactured by Leica camera
Sourced in Germany
COL1A1 is a laboratory equipment product designed for use in scientific research. It serves as a tool for analyzing and quantifying the expression of the COL1A1 gene, which encodes the alpha-1 chain of type I collagen, a major structural protein found in the extracellular matrix. The core function of COL1A1 is to provide researchers with a means to study the regulation and expression of this important genetic marker.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence conjugated secondary antibody, by Beyotime (4 mentions)
Eu5888, by Leica camera (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Keygen Biotech (3 mentions)
Col1a1, by Abcam (2 mentions)
Fluorescence microscope, by Leica camera (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Anti runx2, by Cell Signaling Technology (2 mentions)
Anti foxa2, by Cell Signaling Technology (1 mentions)
P erk, by Leica camera (1 mentions)
T erk, by Cell Signaling Technology (1 mentions)
Runx2, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Col1a1, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Fluorescence microscopy, by Leica camera (1 mentions)
Anti p β catenin, by Cell Signaling Technology (1 mentions)
Anti col1a1, by Abcam (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
4 protocols using col1a1
1
Osteogenesis Induction in BMSCs
2
Immunofluorescence Profiling of Osteogenic Markers
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Cells were cultured in a 12-well plate, and FOXA2, RUNX2, COL1A1, t-ERK and p-ERK were detected using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany). Briefly, cells were fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature, permeabilized and blocked for 30 min in 0.05% Triton X-100 and 5% bovine serum albumin (BSA). Fixed cells were washed three times with PBS and incubated at 4 °C overnight with anti-FOXA2 (1:400; Cell Signalling Technology), RUNX2 (1:1600; Cell Signalling Technology), COL1A1 (1:500; Abcam, Shanghai, China), t-ERK (1:800; Cell Signalling Technology) or p-ERK (1:200; Cell Signalling Technology). Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) at room temperature for 2 h and nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 4 min. Samples were then observed and photographed under a fluorescence microscope (Leica).
3
Osteogenic Differentiation Marker Analysis
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Cells were cultured in induction medium in a 12-well plate, and RUNX2, COL1A1, and β-catenin were detected using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany). Briefly, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized, and blocked for 30 min in 0.05% Triton X-100 and 2% bovine serum albumin. Fixed cells were washed and incubated overnight with anti-RUNX2 (1:1600; Cell Signaling Technology, Shanghai, China), COL1A1 (1:100; Santa Cruz Biotechnology, Shanghai, China), or β-catenin (1:100; Cell Signaling Technology). Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) for 120 min, and nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 2 min. Samples were observed under a fluorescence microscope (Leica).
4
DHA Enhances Osteogenic Markers
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The cells had been cultured with OIM and 1 μM DHA for 72 hours before being seeded within a 12-well plate. Fluorescence microscopy (Leica, Wetzlar, Germany) was used to measure RUNX2, collagen α1 type I (COL1A1), p-ERK, and p-β-catenin. Briefly, cells had been settled at normal temperature within 4% paraformaldehyde for at least 15 min, washed with distilled water three times, blocked in 5% bovine serum albumin as well as 0.03% Triton X-100 for half an hour, washed with distilled water three times, and incubated overnight with anti-p-β-catenin (1:1500, Cell Signaling Technology), anti-p-ERK (1:500, Cell Signaling Technology), anti-COL1A1 (1:500, Abcam, Cambridge, UK), or anti-RUNX2 (1:1,600, Cell Signaling Technology). Cells had been incubated subsequently with a fluorescence-conjugated secondary antibody (Beyotime) for an hour. Cell nuclei had been stained with 4′, 6-diamidino-2-phenylindole for 5 minutes. Cell immunofluorescence was noticed adopting Fluorescence microscopy (Leica).
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