blue alkaline phosphatase alp substrate kit  (Vector Laboratories)

Journal: The FASEB Journal

Article Title: Maternal Exercise Rescues Fetal Akinesia‐Impaired Joint and Bone Development

doi: 10.1096/fj.202503192R

Figure Lengend Snippet: Maternal exercise alters growth plate morphometry and bone neovascularization. (A) Fluorescent staining for collagen type 10 alpha 1 chain (Col10a1) in the proximal hypertrophic zone (HZ) of embryonic day 17.5 (E17.5) humeri. Scale bar = 500 μm. (B) HZ length. (C) Fluorescent staining for alkaline phosphatase (ALP) activity in E17.5 humeri. Scale bar = 250 μm. (D) Primary ossification center (POC) ALP activity and (E) bone collar (BC) ALP activity. (F) Fluorescent staining for blood vessels (endomucin) in E17.5 humeri. Scale bar = 250 μm. (G) Medullary vessel area, (H) Medullary vessel density, (I) BC vessel area, (J) BC vessel density, (K) metaphyseal vessel area, and (L) metaphyseal vessel density. Fetuses from the same litter are marked by the same color datapoint. * p < 0.05 using a Student's t ‐test.

Article Snippet: Alkaline phosphatase (ALP) staining was conducted using the Vector blue Alkaline Phosphatase (ALP) substrate kit (SK‐5300, Vector Laboratories, Newark, CA, USA) according to the manufacturer's instructions.

Techniques: Staining, Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: LRP5-Mediated Lipid Uptake Modulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells

doi: 10.3389/fcell.2021.766815

Figure Lengend Snippet: Lipid scarcity suppressed bone marrow mesenchymal stromal cell (BMSC) osteogenesis and Wnt/beta-catenin signaling activation. (A) Alkaline phosphatase (ALP) staining of BMSCs 7 days after osteogenic induction in culture medium with normal fetal bovine serum (FBS; 10%), lipid-reduced serum (LRS; 10%), SD, and LRS (10%) with 75 μM oleate treatment. (B) ALP activity of BMSCs 5, 7, and 11 days after osteogenic induction in different culture mediums. (C) Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays revealed significantly decreased cell viability in SD treatment BMSCs compared to FBS treatment group, while the cell viability was not significantly changed in LRS and LRS + OL group. (D,E) Western blots showed that the protein expression levels of osteogenic markers Type I Collagen (COL1) and RUNX Family Transcription Factor 2 (RUNX2) and beta-catenin were significantly decreased after lipid deprivation and partially restored by additional oleate treatment 7 days after induction. (F) qRT-PCR revealed that the mRNA expression levels of osteogenic genes, Alpl , Sp7 , and Col1a1 , as well as Wnt target genes Tcf1 , Lef1 , and Axin2 , were significantly decreased after lipid deprivation and partially restored by additional oleate treatment 7 days after induction, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: After 5/7/11 days of osteogenic induction, the osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining kit (VectorLab) in the 12-well plate.

Techniques: Activation Assay, Staining, Activity Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: LRP5-Mediated Lipid Uptake Modulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells

doi: 10.3389/fcell.2021.766815

Figure Lengend Snippet: The suppressed osteogenesis was rescued by oleate supplementation in a dosage-dependent manner. (A) Alkaline phosphatase (ALP) staining of bone marrow mesenchymal stromal cells (BMSCs) 7 days after osteogenic induction in culture medium with normal fetal bovine serum (FBS; 10%), lipid-reduced serum (LRS; 10%), and LRS (10%) with increasing dosages of oleate (25, 50, 75, 100 μM). (B) ALP activity of BMSCs 5, 7, and 11 days after osteogenic induction in different culture mediums. (C,D) Western blots showed that the suppressed protein expression levels of osteogenic markers COL1 and RUNX2 and beta-catenin by lipid deprivation were restored by additional oleate treatment in a dosage-dependent manner. (E) qRT-PCR revealed that the suppressed mRNA expression levels of osteogenic genes Alpl , Sp7 , and Col1a1 and Wnt target genes Tcf1 , Lef1 , and Axin2 by lipid deprivation were restored by additional oleate treatment in a dosage-dependent manner, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: After 5/7/11 days of osteogenic induction, the osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining kit (VectorLab) in the 12-well plate.

Techniques: Staining, Activity Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: Frontiers in Cell and Developmental Biology

Article Title: LRP5-Mediated Lipid Uptake Modulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells

doi: 10.3389/fcell.2021.766815

Figure Lengend Snippet: Low-density lipoprotein receptor-related protein 5 (Lrp5 ) ablation in bone marrow mesenchymal stromal cells (BMSCs) impairs lipid uptake and osteogenesis. (A) BODIPY TM staining revealed significantly decreased lipid droplets in BMSC cytoplasm after Lrp5 ablation using adenoviral Cre-recombinase. (B) Quantified fluorescence intensity of lipid droplets in BMSC cytoplasm indicating impaired lipid uptake of Lrp5 -KO BMSCs. (C) LRP5 protein expression in BMSCs harvested from postnatal day 0 (P0) control mice, conditional knockout (CKO) mice, Lrp5 -control BMSC, and Lrp5 -KO BMSCs. (D) qRT-PCR validated the knockout effect of adenoviral-Cre infection on Lrp5 fl/fl BMSCs. (E) Alkaline phosphatase (ALP) staining of control and Lrp5 -KO BMSCs 7 days after osteogenic induction in culture medium with normal fetal bovine serum (FBS), lipid-reduced serum (LRS), LRS with 100 μM oleate treatment, and LRS with recombinant 100 ng/ml Wnt3a treatment, respectively. (F) ALP activity of BMSCs 7 days after osteogenic induction in different culture mediums. (G) mRNA expression levels in control and Lrp5 -KO BMSCs of osteogenic genes Alpl , Sp7 , and Col1a1 and Wnt target genes Tcf1 , Lef1 , and Axin2 after 7 days of osteogenic induction in FBS or LRS culture medium. (H) mRNA expression levels in control and Lrp5 -KO BMSCs of osteogenic genes Alpl , Sp7 , and Col1a1 as well as Wnt target genes Tcf1 , Lef1 , and Axin2 after 7 days of induction in LRS culture medium with or without Wnt3a treatment, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: After 5/7/11 days of osteogenic induction, the osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining kit (VectorLab) in the 12-well plate.

Techniques: Staining, Fluorescence, Expressing, Knock-Out, Quantitative RT-PCR, Infection, Recombinant, Activity Assay