mouse anti hbcl2  (Novus Biologicals)

Journal: Scientific reports

Article Title: hBcl2 overexpression in BMSCs enhances resistance to myelin debris-induced apoptosis and facilitates neuroprotection after spinal cord injury in rats.

doi: 10.1038/s41598-024-52167-4

Figure Lengend Snippet: Figure 2. Expression of hBcl2 in different groups of BMSCs models. (A) Cellular immunofluorescence staining was used to determine the amounts of hBcl2 protein (red) and GFP protein (green) expression in cells from the hBcl2, cb, NC, and normal BMSCs groups. (B) Quantitative analysis of the average fluorescence intensity of hBcl2 expression in each group (At least three independent replicates were performed for each experiment. Data are presented as mean ± SD, ****P < 0.0001 vs. hBcl2 group analyzed by one-way ANOVA, followed by Tukey’s post hoc test). (C) Western blot results revealed hBcl2 protein expression in the hBcl2, cb, NC, and BMSCs groups (original blots were presented in Supplementary Fig. 1). Scale bar = 10 μm (A).

Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.5% Tween-20 (TBST) at room temperature for 30 min. After washing the membranes with TBST, they were incubated overnight at 4 °C with mouse anti-βTubulin (1:10,000, T0023, Affinity, USA) and mouse anti-hBcl2 antibody (1:100, NBP2-15200, Novus, USA).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot

Journal: Scientific reports

Article Title: hBcl2 overexpression in BMSCs enhances resistance to myelin debris-induced apoptosis and facilitates neuroprotection after spinal cord injury in rats.

doi: 10.1038/s41598-024-52167-4

Figure Lengend Snippet: Figure 3. Overexpression of hBcl2 protein can enhance the anti-apoptotic ability of BMSCs in vitro. (A) The CCK8 method was used to detect cell proliferation in hBcl2, cb, NC, and BMSCs groups at 1, 2, 3, 4, 5, 6, and 7 days (At least three independent replicates were performed for each experiment. hBcl2 comparisons are shown in red, cb comparisons are shown in gold, BMSC comparisons are shown in black, and NC comparisons are shown in blue. Data are presented as mean ± SD, ***P < 0.001, **P < 0.01, *P < 0.05 vs. BMSC group; ###P < 0.001, ##P < 0.01 vs. NC group, analyzed by two-way ANOVA, followed by Tukey’s post hoc test). (B) The CCK8 method was used to detect cell survival in each group at 1, 2, 3, 4, 5, 6, 7 and 8 days by using serum deprivation treatment (At least three independent replicates were performed for each experiment. hBcl2 comparisons are shown in red, cb comparisons are shown in gold, BMSC comparisons are shown in black, and NC comparisons are shown in blue. Data are presented as mean ± SD, **P < 0.01, *P < 0.05, vs. BMSC group; ###P < 0.001, ##P < 0.01, #P < 0.05 vs. NC group, analyzed by two-way ANOVA, followed by Tukey’s post hoc test). (C) The apoptosis of cells in each group was measured by TUNEL staining. DAPI (blue) stained nuclei indicating the total number of cells, and TUNEL (red) indicates the apoptotic cells. (D) The proportion of TUNEL positive cells in each group (At least three independent replicates were performed for each experiment. ****P < 0.0001 vs. hBcl2 group, analyzed by one-way ANOVA, followed by Tukey’s post hoc test). Scale bar = 50 μm.

Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.5% Tween-20 (TBST) at room temperature for 30 min. After washing the membranes with TBST, they were incubated overnight at 4 °C with mouse anti-βTubulin (1:10,000, T0023, Affinity, USA) and mouse anti-hBcl2 antibody (1:100, NBP2-15200, Novus, USA).

Techniques: Over Expression, In Vitro, TUNEL Assay, Staining

Journal: Scientific reports

Article Title: hBcl2 overexpression in BMSCs enhances resistance to myelin debris-induced apoptosis and facilitates neuroprotection after spinal cord injury in rats.

doi: 10.1038/s41598-024-52167-4

Figure Lengend Snippet: Figure 4. hBcl2 overexpressing BMSCs with enhanced anti-apoptotic ability could resist myelin debris-induced early apoptosis in vitro. (A) Cellular immunofluorescence staining of cleaved caspase3 was used to assess apoptosis in the hBcl2, cb, NC, and BMSCs groups after incubating cells with macrophage conditioned medium (Mφ CM) for 24 h. White arrows represent apoptotic cells. (B) Cleaved caspase3 immunofluorescence staining was used to determine the apoptosis of cells in each group after 24 h cell culture in Dil-myelin CM (500 μg/ mL). (C,D) Quantitative analysis of the proportion of cleaved caspase3 positive cells in the total number of cells, which were culture in macrophage CM (C) or myelin debris (D) (At least three independent replicates were performed for each experiment. Data are presented as mean ± SD, ****P < 0.0001 vs. hBcl2 group, cb vs. NC group, analyzed by one-way ANOVA, followed by Tukey’s post hoc test). (E) The morphological changes of apoptotic cells (black arrow) were observed under a light microscope after 24 h cell culture in myelin debris (500 μg/mL). Scale bars = 50 μm (A,B,E).

Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.5% Tween-20 (TBST) at room temperature for 30 min. After washing the membranes with TBST, they were incubated overnight at 4 °C with mouse anti-βTubulin (1:10,000, T0023, Affinity, USA) and mouse anti-hBcl2 antibody (1:100, NBP2-15200, Novus, USA).

Techniques: In Vitro, Immunofluorescence, Staining, Cell Culture, Light Microscopy