rabbit anti type 3 collagen  (Proteintech)

Journal: Molecular Medicine

Article Title: Osthole ameliorates wear particle-induced osteogenic impairment by mitigating endoplasmic reticulum stress via PERK signaling cascade

doi: 10.1186/s10020-024-01034-z

Figure Lengend Snippet: OST treatment attenuated TiPs-induced osteogenesis inhibition in vivo. ( A ) Representative Masson’s trichrome staining images of calvarial sections. Scale bar, 200 μm (upper), 50 μm (lower). ( B ) Quantitative analysis of new bone area fraction (%) determined by Masson’s trichrome staining. ( C ) Representative images of calcein (green) and alizarin red (red) double labeling with a 10-day interval. Scale bar, 50 μm (upper), 20 μm (lower). ( D ) Quantitative analysis of average periosteum mineral apposition rates (MAR, µm/d). ( E-F ) Representative images and quantitative analysis of immunohistochemical (IHC) staining for osteocalcin (OCN). Scale bar, 100 μm (upper), 50 μm (lower). ( G-H ) Representative images and quantitative analysis of IHC staining for collagen type I alpha 1 (COL1α1). Scale bar, 100 μm (upper), 50 μm (lower). ( I-J ) Western blot analysis of the expression of osteogenic markers (OCN, COL1α1) in calvarial bone tissue samples from each group. n = 6. The selected images reflect typical examples from each group, closely representing the median degree as per statistical analysis. Data are presented as mean ± SD. One-way ANOVA with Tukey’s post hoc test. The relative COL1α1 protein levels were analyzed using Brown-Forsythe and Welch ANOVA tests followed by Tamhane T2 post hoc tests. ** P < 0.01 versus the Sham group. # P < 0.05 and ## P < 0.01 versus the TiPs group. ns , not statistically significant versus the TiPs group

Article Snippet: The primary antibodies used for the IHC assay were as follows: anti-osteocalcin (OCN) rabbit IgG (Proteintech, 23418-1-AP), anti-Collagen Type I Alpha 1 (COL1α1) rabbit IgG (Boster, PB0981), anti-GRP78 rabbit IgG (Abcam, ab21685), anti-CHOP rabbit IgG (Affinity, AF6277), anti-OCN mouse IgG (Santa Cruz Biotechnology, sc-390877), anti-C-CASP3 rabbit IgG (Cell Signaling Technology, 9664), anti-phospho-PERK (p-PERK) rabbit IgG (Affinity, DF7576), anti-Phospho-eIF2 alpha (p-eIF2α) rabbit IgG (Affinity, AF3087), anti-ATF4 (Affinity, DF6008).

Techniques: Inhibition, In Vivo, Staining, Labeling, Immunohistochemical staining, Immunohistochemistry, Western Blot, Expressing

Journal: Molecular Medicine

Article Title: Osthole ameliorates wear particle-induced osteogenic impairment by mitigating endoplasmic reticulum stress via PERK signaling cascade

doi: 10.1186/s10020-024-01034-z

Figure Lengend Snippet: OST treatment improved the impaired osteogenic potential of osteoblasts upon TiPs exposure in vitro. ( A ) Heat map of the osteogenesis-related DEGs in the TiPs group versus the Control group. ( B ) GSEA illustrating the enrichment of osteoblast differentiation and bone mineralization GO items, which are negatively regulated in the TiPs group compared to the Control group. ( C ) Representative images of alkaline phosphatase (ALP) staining of osteoblasts treated as intended following 7 days of osteogenic induction. Scale bar, 5 mm (upper), 500 μm (lower). ( D ) Quantitative analysis of relative ALP activity. ( E ) Representative images of alizarin red S (ARS) staining of osteoblasts treated as intended following 14 days of osteogenic induction. Scale bar, 5 mm (upper), 500 μm (lower). ( F ) Quantitative analysis of ARS staining. ( G-H ) Western blot analysis of the expression of osteogenesis-related proteins (COL1α1, ALP, RUNX2, and OCN) in osteoblasts treated as intended following 3 days of osteogenic induction. n = 3. The selected images reflect typical examples from each group, closely representing the median degree as per statistical analysis. Data are presented as mean ± SD. One-way ANOVA with Tukey’s post hoc test. ** P < 0.01 versus the Control group. # P < 0.05 and ## P < 0.01 versus the TiPs group. ns , not statistically significant versus the TiPs group

Article Snippet: The primary antibodies used for the IHC assay were as follows: anti-osteocalcin (OCN) rabbit IgG (Proteintech, 23418-1-AP), anti-Collagen Type I Alpha 1 (COL1α1) rabbit IgG (Boster, PB0981), anti-GRP78 rabbit IgG (Abcam, ab21685), anti-CHOP rabbit IgG (Affinity, AF6277), anti-OCN mouse IgG (Santa Cruz Biotechnology, sc-390877), anti-C-CASP3 rabbit IgG (Cell Signaling Technology, 9664), anti-phospho-PERK (p-PERK) rabbit IgG (Affinity, DF7576), anti-Phospho-eIF2 alpha (p-eIF2α) rabbit IgG (Affinity, AF3087), anti-ATF4 (Affinity, DF6008).

Techniques: In Vitro, Control, Staining, Activity Assay, Western Blot, Expressing

Journal: Molecular Medicine

Article Title: Osthole ameliorates wear particle-induced osteogenic impairment by mitigating endoplasmic reticulum stress via PERK signaling cascade

doi: 10.1186/s10020-024-01034-z

Figure Lengend Snippet: The selective PERK agonist CCT020312 abrogated the protective effects of OST against TiPs-induced osteogenic impairment in vitro. ( A ) Western blots showing the protein levels of PERK, p-PERK, eIF2α, p-eIF2α, and ATF4 in osteoblasts exposed TiPs (50 µg/ml) following treatment with OST (20 µM) or OST combined with CCT020312 (10 µM) for 24 h. ( B ) Representative images of GRP78 immunofluorescence staining (green) in osteoblasts treated as intended. Scale bar, 100 μm. ( C ) Representative images of CHOP immunofluorescence staining (red) in osteoblasts treated as intended. Scale bar, 100 μm. ( D ) Representative images of C-CASP3 immunofluorescence staining (green) in osteoblasts treated as intended, with F-actin labeled in red using phalloidin. Scale bar, 100 μm. ( E ) Western blots showing the protein levels of GRP78, CHOP, and C-CASP3 in osteoblasts treated as intended. ( F ) Representative images of TUNEL staining in osteoblasts treated as intended. Scale bar, 100 μm. ( G-H ) Flow cytometry analysis of apoptosis levels in osteoblasts treated as intended, using Annexin V/PI staining for detection. ( I ) Quantitative analysis of the TUNEL staining results of (F). ( J ) Western blot analysis of the expression of osteogenesis-related proteins (COL1α1, ALP, RUNX2, and OCN) in osteoblasts treated as intended following 3 days of osteogenic induction. ( K ) Representative images of ALP staining of osteoblasts treated as intended following 7 days of osteogenic induction. Scale bar, 5 mm (upper), 500 μm (lower). ( L ) Representative images of ARS staining of osteoblasts treated as intended following 14 days of osteogenic induction. Scale bar, 5 mm (upper), 500 μm (lower). n = 3. The selected images reflect typical examples from each group, closely representing the median degree as per statistical analysis. Data are presented as mean ± SD. One-way ANOVA with Tukey’s post hoc test. ** P < 0.01 versus the Control group. ## P < 0.01 versus the TiPs group. && P < 0.01 versus the TiPs + OST group

Article Snippet: The primary antibodies used for the IHC assay were as follows: anti-osteocalcin (OCN) rabbit IgG (Proteintech, 23418-1-AP), anti-Collagen Type I Alpha 1 (COL1α1) rabbit IgG (Boster, PB0981), anti-GRP78 rabbit IgG (Abcam, ab21685), anti-CHOP rabbit IgG (Affinity, AF6277), anti-OCN mouse IgG (Santa Cruz Biotechnology, sc-390877), anti-C-CASP3 rabbit IgG (Cell Signaling Technology, 9664), anti-phospho-PERK (p-PERK) rabbit IgG (Affinity, DF7576), anti-Phospho-eIF2 alpha (p-eIF2α) rabbit IgG (Affinity, AF3087), anti-ATF4 (Affinity, DF6008).

Techniques: In Vitro, Western Blot, Immunofluorescence, Staining, Labeling, TUNEL Assay, Flow Cytometry, Expressing, Control