immunofluorescence staining for inos, cd206, cd31, and α-sma gb111364-100  (Servicebio Inc)

Journal: Fluids and Barriers of the CNS

Article Title: Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage

doi: 10.1186/s12987-024-00550-8

Figure Lengend Snippet: Schematic of the experimental design and subsequent analyses. IF indicates immunofluorescence staining; TEM indicates transmission electron microscopy; i.v. indicates intravenous injection; WB indicates Western blot; and EB indicates the Evans blue experiment

Article Snippet: The endothelial cells were passaged 3 times to purify the endothelial cells, and the cells were used for subsequent cell experiments after the purity of the cells was > 99%, as determined by immunofluorescence staining with CD 31 (1:100, NB100-2284, NOVUS).

Techniques: Immunofluorescence, Staining, Transmission Assay, Electron Microscopy, Injection, Western Blot

Journal: Fluids and Barriers of the CNS

Article Title: Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage

doi: 10.1186/s12987-024-00550-8

Figure Lengend Snippet: BBB disruption and cerebral edema after SAH. A TEM of cerebral microvasculature in the cortical region of C57BL/6 mice on days 1, 3 and 7 in the sham and SAH groups (scale bar, 1 μm). (A-astrocytes end-foot; E-endothelial cells; white arrows-gap junctions; yellow arrows-tight junctions; red arrows -vascular basement membrane; *-blood vessels; PMNn-polymorphonuclear neutrophils). B and D Immunofluorescence labeling of ZO1 (Zonula occludens protein 1, red), CD31 (platelet endothelial cell adhesion molecule-1, green), and DAPI (4ʹ,6-diamidino-2-phenylindole, blue) on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice (scale bar, 50 μm). n = 6 per group; one-way ANOVA, **P < 0.01 vs. the sham group; ****P < 0.0001 vs. the sham group. C, E and F Representative images of Evans blue (red) autofluorescence and Evans blue extravasation on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice (scale bar, 50 μm). n = 6 per group; one-way ANOVA; ****P < 0.0001 vs. the sham group. G Brain water content was measured on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice. n = 8/7, one-way ANOVA, **P < 0.01 vs. the sham group; ****P < 0.0001 vs. the sham group

Article Snippet: The endothelial cells were passaged 3 times to purify the endothelial cells, and the cells were used for subsequent cell experiments after the purity of the cells was > 99%, as determined by immunofluorescence staining with CD 31 (1:100, NB100-2284, NOVUS).

Techniques: Disruption, Membrane, Immunofluorescence, Labeling

Journal: Fluids and Barriers of the CNS

Article Title: Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage

doi: 10.1186/s12987-024-00550-8

Figure Lengend Snippet: Spatial changes and the localization of GL structures after SAH. A and C Immunofluorescence labeling of CLDN1 (claudin-1, red), CX43 (connexin 43, green), GFAP (glial fibrillary acidic protein, white), and DAPI (blue) on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice (scale bar, 50 μm). CLDN1 colocalized with GFAP. n = 6 per group; one-way ANOVA; ****P < 0.0001 vs. the sham group. B and D Immunofluorescence labeling of CLDN1 (red), CX43 (green), GFAP (white) and DAPI (blue) after the treatment of primary astrocytes with OGD and OxyHb (scale bar, 50 μm). n = 6 per group; one-way ANOVA; ****P < 0.0001 vs. the astrocyte vehicle group. E TEER values of the astrocyte Vehicle group, the OGD-treated group and the OxyHb intervention group after 6 h, 12 h, 24 h and 48 h. n = 6 per group, 1-way ANOVA, **P < 0.01 vs. the ast vehicle group; ***P < 0.001 vs. the ast vehicle group; ****P < 0.0001 vs. the ast vehicle group

Article Snippet: The endothelial cells were passaged 3 times to purify the endothelial cells, and the cells were used for subsequent cell experiments after the purity of the cells was > 99%, as determined by immunofluorescence staining with CD 31 (1:100, NB100-2284, NOVUS).

Techniques: Immunofluorescence, Labeling

Journal: Fluids and Barriers of the CNS

Article Title: Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage

doi: 10.1186/s12987-024-00550-8

Figure Lengend Snippet: GL barrier function and the effect on neurological outcomes after SAH. A and D Immunofluorescence labeling of CD45 (leukocyte common antigen, red), CD31 (green), GFAP (white) and DAPI (blue) on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice (scale bar, 50 μm). n = 6 per group; one-way ANOVA, *P < 0.05 vs. the sham group; ***P < 0.001 vs. the sham group; ****P < 0.0001 vs. the sham group. B and E Flow cytometry plots and quantification showing the expression of infiltrating inflammatory cells, including NK cells and neutrophils, obtained from brain tissue on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice (scale bar, 50 μm). n = 6 per group; one-way ANOVA, **P < 0.01 vs. the sham group; ***P < 0.001 vs. the sham group; ****P < 0.0001 vs. the sham group. C and F Open field test. The movement trajectories and the quantitative statistics of distances on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice. The mice underwent the training test on the open field 24 h before the test. n = 8; one-way ANOVA, ***P < 0.001 vs. the sham group; ****P < 0.0001 vs. the sham group. G and H Modified Garcia scores ( G ) and beam balance scores ( H ) were measured on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice. n = 8/7, one-way ANOVA, **P < 0.01 vs. the sham group; ****P < 0.0001 vs. the sham group

Article Snippet: The endothelial cells were passaged 3 times to purify the endothelial cells, and the cells were used for subsequent cell experiments after the purity of the cells was > 99%, as determined by immunofluorescence staining with CD 31 (1:100, NB100-2284, NOVUS).

Techniques: Immunofluorescence, Labeling, Flow Cytometry, Expressing, Modification

Journal: Fluids and Barriers of the CNS

Article Title: Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage

doi: 10.1186/s12987-024-00550-8

Figure Lengend Snippet: Changes in EGLN3 expression in endothelial cells after SAH. A and C Immunofluorescence labeling of EGLN3 (prolyl hydroxylase EGLN3, green), CD31 (red), and DAPI (blue) on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice (scale bar, 50 μm). n = 6 per group; one-way ANOVA; ****P < 0.0001 vs. the sham group. B and D Immunofluorescence labeling of EGLN3 (green), vWF (von Willebrand factor, white), and DAPI (blue) in the endothelial cell vehicle group, the OxyHb intervention group and the OGD-treated group after 24 h. n = 6 per group, 1-way ANOVA, ****P < 0.0001 vs. the Endo vehicle group. E and F Western blotting and quantitative analysis of Hif1-alpha on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice and the endothelial cell vehicle group, the OxyHb intervention group and the OGD-treated group after 24 h. n = 3 per group, 1-way ANOVA, ****P < 0.0001 vs. the sham group

Article Snippet: The endothelial cells were passaged 3 times to purify the endothelial cells, and the cells were used for subsequent cell experiments after the purity of the cells was > 99%, as determined by immunofluorescence staining with CD 31 (1:100, NB100-2284, NOVUS).

Techniques: Expressing, Immunofluorescence, Labeling, Western Blot

Journal: Fluids and Barriers of the CNS

Article Title: Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage

doi: 10.1186/s12987-024-00550-8

Figure Lengend Snippet: Changes in PKM2 expression in astrocytes after SAH. A and C Immunofluorescence labeling of PKM2 (pyruvate kinase M2, green), GFAP (red), and DAPI (blue) on days 1, 3 and 7 in the sham and SAH groups of C57BL/6 mice (scale bar, 50 μm). n = 6 per group; one-way ANOVA, **P < 0.01 vs. the sham group; ****P < 0.0001 vs. the sham group. B and D Immunofluorescence labeling of PKM2 (green), CLDN1 (red), GFAP (white), and DAPI (blue) in the astrocyte vehicle group, the astrocyte OGD group, the ast-endo medium group, the ast-endo OGD medium group and the ast-endo OxyHb medium group after 24 h. n = 6 per group, 1-way ANOVA, ****P < 0.0001 vs. the ast vehicle group

Article Snippet: The endothelial cells were passaged 3 times to purify the endothelial cells, and the cells were used for subsequent cell experiments after the purity of the cells was > 99%, as determined by immunofluorescence staining with CD 31 (1:100, NB100-2284, NOVUS).

Techniques: Expressing, Immunofluorescence, Labeling

Journal: Fluids and Barriers of the CNS

Article Title: Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage

doi: 10.1186/s12987-024-00550-8

Figure Lengend Snippet: Changes in EGLN3 in astrocytes after SAH and the correlation between EGLN3 and PKM2. A and B Immunofluorescence labeling of CLDN1 (red), EGLN3 (green), GFAP (white), and DAPI (blue) in the ast-endo medium group, the ast-endo OGD medium group and the ast-endo OxyHb medium group after 24 h. n = 6 per group, 1-way ANOVA, ****P < 0.0001 vs. Ast Vehicle group. C and D Western blotting and quantitative analysis of EGLN3 in the cell supernatants of the vehicle group, the OGD-treated group and the OxyHb intervention group after 24 h. n = 3 per group, 1-way ANOVA, ****P < 0.0001 vs. the sham group. E The results of process docking, the three-dimensional structure of PKM2 (yellow) and EGLN3 (blue) process docking and docking sites. F The protein interactions were analyzed by coimmunoprophylaxis. The interaction between EGLN3 and PKM2 was verified by forward and reverse experiments

Article Snippet: The endothelial cells were passaged 3 times to purify the endothelial cells, and the cells were used for subsequent cell experiments after the purity of the cells was > 99%, as determined by immunofluorescence staining with CD 31 (1:100, NB100-2284, NOVUS).

Techniques: Immunofluorescence, Labeling, Western Blot

Journal: Fluids and Barriers of the CNS

Article Title: Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage

doi: 10.1186/s12987-024-00550-8

Figure Lengend Snippet: EGLN3 or PKM2 could enhance the tight junction expression of GL in vivo. A–D Immunofluorescence labeling of CLDN1 (red), EGLN3 (green), GFAP (white) and DAPI (blue) on days 1, 3 and 7 in the sham and SAH groups of WT mice and EGLN3 CKI/CKI, Cdh5−creERT2 mice; Immunofluorescence labeling of CLDN1 (red), PKM2 (green), GFAP (white) and DAPI (blue) on days 1, 3 and 7 in the sham and SAH groups of WT mice, PKM2 CKI/CKI, Aldh1| 1−creERT2 mice and Shikonin-treated EGLN3 CKI/CKI, Cdh5−creERT2 mice (scale bar, 50 μm). n = 6 per group; two-way ANOVA, ***P < 0.001; ****P < 0.0001

Article Snippet: The endothelial cells were passaged 3 times to purify the endothelial cells, and the cells were used for subsequent cell experiments after the purity of the cells was > 99%, as determined by immunofluorescence staining with CD 31 (1:100, NB100-2284, NOVUS).

Techniques: Expressing, In Vivo, Immunofluorescence, Labeling

Journal: Fluids and Barriers of the CNS

Article Title: Endothelial EGLN3-PKM2 signaling induces the formation of acute astrocytic barrier to alleviate immune cell infiltration after subarachnoid hemorrhage

doi: 10.1186/s12987-024-00550-8

Figure Lengend Snippet: EGLN3 or PKM2 could enhance the tight junction expression of GL in vitro. A–D Immunofluorescence labeling of PKM2 (green), CLDN1 (red), and GFAP (white) and DAPI (blue). The following four types of cells were used: the vehicle group (astrocytes were not treated); the EGLN3(+) group (endothelial cells were transfected with a pcDNA3.1(+)-EGLN3 plasmid); the PKM2(+) group (astrocytes were transfected with a pcDNA3.1(+)-PKM2 plasmid); and the EGLN3(+) PKM2(−) group (astrocytes were transfected with a Psd1211-U6-shRNA-PKM2 plasmid, and endothelial cells were transfected with a pcDNA3.1(+)-EGLN3 plasmid). The cells were treated in the following four different ways: astrocytes were not treated, astrocytes were cultured with endothelial cell culture medium, astrocytes were cultured with OGD-treated endothelial cell culture medium, and astrocytes were cultured with OxyHb-treated endothelial cell culture medium. Immunofluorescence staining was performed 24 h later. n = 6 per group; two-way ANOVA; ****P < 0.0001. E TEER values were measured. The following three types of cells were used: the WT group (astrocytes were not treated), the pcDNA3.1(+)-EGLN3 endothelial group (endothelial cells were transfected with a pcDNA3.1(+)-EGLN3 plasmid) and the pcDNA3.1(+)-PKM2 astrocyte group (astrocytes were transfected with a pcDNA3.1(+)-PKM2 plasmid). The cells were treated in the following two different ways: astrocytes were cultured with OGD-treated endothelial cell culture medium, and astrocytes were cultured with OxyHb-treated endothelial cell culture medium for 0 h, 6 h, 12 h, 24 h or 48 h. n = 6 per group, 2-way ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: The endothelial cells were passaged 3 times to purify the endothelial cells, and the cells were used for subsequent cell experiments after the purity of the cells was > 99%, as determined by immunofluorescence staining with CD 31 (1:100, NB100-2284, NOVUS).

Techniques: Expressing, In Vitro, Immunofluorescence, Labeling, Transfection, Plasmid Preparation, shRNA, Cell Culture, Staining

Journal: Materials Today Bio

Article Title: Multifunctional hydrogel promotes rotator cuff healing through anti-inflammation and vascularization

doi: 10.1016/j.mtbio.2025.102016

Figure Lengend Snippet: Evaluation of the vascularization potential of the LA-CMCS-OHA hydrogel in vitro . (A, B) VEGFA protein levels were analyzed via western blot and semi-quantification. (C) VEGF levels were quantitatively measured using ELISA. (D, E) HUVEC proliferation rates were assessed through EDU analysis of CMCS-OHA and LA-CMCS-OHA hydrogels. scale bar: 10 μm. (F, G) CD31 protein levels were analyzed using immunofluorescence and semi-quantification. scale bar: 10 μm. (H) CD31 mRNA levels were determined via RT-qPCR. (I–L) Tube formation assays were conducted at 3 and 5 h to evaluate differences in tube formation, total length, and the number of junctions among groups. scale bar: 10 μm.

Article Snippet: Coronal sections were prepared for immunological examination, including immunofluorescence staining for iNOS, CD206, CD31, and α-SMA (Servicebio, GB111364-100, 1:500); immunohistochemistry for IL-6, TNF-α, and IL-10; and histopathological examination using H&E, Toluidine blue, and Masson's trichrome staining.

Techniques: In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Quantitative RT-PCR