recombinant human wnt2 protein  (Bio-Techne corporation)

Journal: Advanced Science

Article Title: Optogenetic Stimulation of mPFC Alleviates White Matter Injury‐Related Cognitive Decline after Chronic Ischemia through Adaptive Myelination

doi: 10.1002/advs.202202976

Figure Lengend Snippet: Activation of glutamatergic neurons in the mPFC upregulates Wnt2 expression in the corpus callosum. a) Heatmap of the results of mPFC tissue microarray analysis between the BCAS mCherry and BCAS ChR2 groups at 2 months after surgery. n = 3 per group. b) Volcano plot of the results of mPFC tissue microarray analysis between the BCAS mCherry and BCAS ChR2 groups at 2 months after surgery (fold change > 2; p < 0.05). The DEGs are listed. n = 3 per group. c) Real‐time PCR for validation of the identified DEGs among the sham mCherry, BCAS mCherry, and BCAS ChR2 groups at 2 months after surgery. n = 7–8 per group. d) Real‐time PCR analysis of Wnt2 expression in the mPFC in the sham mCherry, BCAS mCherry, and BCAS hM3D groups at 2 months after surgery. n = 7–8 per group. e) Real‐time PCR analysis of Wnt2 expression in different types of primary cortical neural cells. n = 3 per group. f) Representative immunostaining of NeuN and Wnt2 in the mPFC in the sham mCherry, BCAS mCherry, and BCAS ChR2 groups at 2 months after surgery. Scale bar: 50 µm. g) Representative immunostaining of NF‐H and Wnt2 in the corpus callosum in the sham mCherry, BCAS mCherry, and BCAS ChR2 groups at 2 months after surgery. Scale bar: 35 µm. Quantification of immunostaining of Wnt2 in the h) mPFC and i) corpus callosum in the sham mCherry, BCAS mCherry, and BCAS ChR2 groups at 2 months after surgery. The fluorescence intensity in each image was normalized to that of NeuN and NF‐H. The measured values were normalized to the mean value of the BCAS group. n = 3 per group. j) Representative immunostaining of NeuN and Wnt2 in the mPFC in the sham mCherry, BCAS mCherry, and BCAS hM3D groups at 2 months after surgery. Scale bar: 50 µm. k) Representative immunostaining of NF‐H and Wnt2 in the corpus callosum in the sham mCherry, BCAS mCherry, and BCAS hM3D groups at 2 months after surgery. Scale bar: 35 µm. Quantification of Wnt2 immunostaining in the l) mPFC and m) corpus callosum in the sham mCherry, BCAS mCherry, and BCAS hM3D groups at 2 months after surgery. The fluorescence intensity in each image was normalized to that of NeuN and NF‐H. The measured values were normalized to the mean value of the BCAS group. n = 3 per group. The data are presented as the mean ± SEM. p ‐values were determined by the Kruskal–Wallis test with Dunn's post‐hoc analysis in (d); by 1‐way ANOVA with Tukey's post‐hoc analysis in (c) and (e); and by Student's t ‐test in (h), (i), (l), and (m). * p < 0.05, ** p < 0.01.

Article Snippet: Afterward, 50 ng mL −1 T3 (Thermo Fisher Scientific, USA), 200 ng mL −1 recombinant Wnt2 protein (Cusabio, China), 100 ng mL −1 recombinant Dkk1 protein (Cusabio, China) and basic medium DMEM F12 supplemented with 2% B27, 100 U mL −1 antibiotics, and 20 ng mL −1 CNTF (Thermo Fisher Scientific, USA) were applied to induce differentiation.

Techniques: Activation Assay, Expressing, Microarray, Real-time Polymerase Chain Reaction, Biomarker Discovery, Immunostaining, Fluorescence

Journal: Advanced Science

Article Title: Optogenetic Stimulation of mPFC Alleviates White Matter Injury‐Related Cognitive Decline after Chronic Ischemia through Adaptive Myelination

doi: 10.1002/advs.202202976

Figure Lengend Snippet: Overexpression of Wnt2 in mPFC glutamatergic neurons alleviates myelin injury and improves cognition. Results of the a) Y‐maze and b) T‐maze tests showing the spontaneous alternation percentage in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 15–16 per group. Results of the open field test showing c) the total distance travelled and d) time spent in the corner area and e) center area in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 15–16 per group. f) Heatmaps generated from DTI axial views of FA acquired from the control and Camk2a‐Wnt2 groups at 2 months after surgery. g) Quantification of FA values in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. The measured values were normalized to the mean value of the control group. n = 11 per group. h) Representative images of Black‐gold staining and immunostaining of MBP and MAG in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. Scale bar: 200 µm. Quantification of i) Black‐gold staining and immunostaining of j) MBP and k) MAG in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 4 per group. l) Representative TEM images of the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. Scale bar: 1 µm. m) The percentage of myelinated axons in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 4 per group. n) Scatterplots of the myelin g‐ratio as a function of the axon diameter in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. Axons were selected from 4 mice per group for measurement. n = 138 and n = 121 measured axons for each group, respectively. o) Representative immunostaining of CC1 and Olig2 in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. Scale bar: 50 µm. Density of p) CC1 + Olig2 + cells and q) CC1 − Olig2 + cells in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 5 per group. r) Proportion of CC1 + /Olig2 + cells in the corpus callosum in the control and Camk2a‐Wnt2 groups at 2 months after surgery. n = 5 per group. s) Immunoblot bands and t) quantification of MBP and u) MAG expression in OPCs in the control, T3, and T3+ rmWnt2 groups. The intensity of each immunoblot band was normalized to that of the β ‐actin band. The measured values were normalized to the mean value of the control group. n = 3 per group. The data are presented as the mean ± SEM. p ‐values were determined by Student's t ‐test in (b–e), (i–k), (m), (p–r), and (t); by the Mann–Whitney test in (a), (g), and (n); and by 1‐way ANOVA with Tukey's post‐hoc analysis in (u). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Afterward, 50 ng mL −1 T3 (Thermo Fisher Scientific, USA), 200 ng mL −1 recombinant Wnt2 protein (Cusabio, China), 100 ng mL −1 recombinant Dkk1 protein (Cusabio, China) and basic medium DMEM F12 supplemented with 2% B27, 100 U mL −1 antibiotics, and 20 ng mL −1 CNTF (Thermo Fisher Scientific, USA) were applied to induce differentiation.

Techniques: Over Expression, Control, Generated, Staining, Immunostaining, Western Blot, Expressing, MANN-WHITNEY