Journal: Scientific Reports

Article Title: Dynamics and function of CXCR4 in formation of the granule cell layer during hippocampal development

doi: 10.1038/s41598-017-05738-7

Figure Lengend Snippet: Altered distribution pattern of Prox1-positive granule cell precursors induced by the CXCR4 antagonist AMD3100. AMD3100 or vehicle (control) was injected into the telencephalic lateral ventricle of E15.5 embryos in utero , and the embryos were left to develop until E18.5 before sampling. ( A , B ) Immunoreactivity for CXCR4 and Prox1, a marker for granule cells in the hippocampus at E18.5. ( A ) In the control, cells strongly positive for Prox1 were mainly localized in the dentate gyrus (DG). ( B ) In AMD3100-treated mice, cells strongly positive for Prox1 were observed not only in the DG (arrowhead), but also in the ventricular zone (VZ) and dentate migratory stream (DMS, arrows). ( C ) The number of Prox1-positive cells in the DG, and in the VZ and DMS. The number of Prox1-positive cells in the VZ and DMS is significantly higher in the AMD3100-treated mice than in control mice (*P < 0.05, Student’s t test). No significant difference was observed in the total number of Prox1-positive cells between the two groups. Ten sections were counted from each of 5 (control) or 6 (AMD3100-treated) animals. F, fimbria; HF, hippocampal fissure; V, ventricle. Scale bar = 50 µm in A and B .

Article Snippet: The primary antibodies used were as follows: anti-CXCR4 UMB2 (1:200, rabbit monoclonal, Abcam), anti-GFP (1:1,000, chick polyclonal, Abcam), anti-GM130 (1:500, mouse monoclonal, BD Transduction Laboratories), anti-γ-tubulin (1:200, mouse monoclonal, Sigma), anti-Ki67 (1:500, rabbit polyclonal, Novocastra), anti-LAMP1 (1:1000, rat monoclonal, Abcam), anti-NeuroD (1:200, goat polyclonal, Santa Cruz Biotechnology, Inc.), anti-Prox1 (1:1,000, goat polyclonal, R&D Systems), anti-p73 (1:200, rabbit polyclonal, Santa Cruz Biotechnology, Inc.).

Techniques: Injection, In Utero, Sampling, Marker

Journal: Scientific Reports

Article Title: Dynamics and function of CXCR4 in formation of the granule cell layer during hippocampal development

doi: 10.1038/s41598-017-05738-7

Figure Lengend Snippet: Ectopic positioning of GCPs in the hippocampal fissure-surrounding region (HFSR) induced by the CXCR4 antagonist AMD3100. AMD3100 or vehicle (control) was injected into the lateral ventricle of E15.5 embryos in utero , and the embryos were left to develop until E18.5. ( A,B ) Expression of Gfap -GFP and p73, a marker for Cajal-Retizus cells in the hippocampus of Gfap -GFP mice. The boxed regions in A1 and B1 are enlarged in A2 and B2, respectively. A3 and B3 are 3-dimensional images of A2 and B2, respectively, that were reconstructed from 10 optical slices. p73-positive cells accumulated in the HFSR in both the control ( A1–3 ) and AMD3100-treated ( B1–3 ) mice. In the control, only a few Gfap -GFP+ cells (arrows) were observed in the HFSR ( A1–3 ), although the processes extending from the Gfap -GFP+ cells in the dentate gyrus (DG, open arrowheads) invaded into the HFSR (white arrowheads). In the AMD3100-injected mice, many Gfap -GFP-positive cells accumulated in the HFSR ( B1–3 , arrows). ( C,D ) The proportion of Gfap- GFP+ cells in the HFSR to those in the HFSR and DG. A significant increase in Gfap -GFP+ cells was observed in the AMD3100-treated mice ( C , *P < 0.05; Student t-test). However, no difference was detected between the groups in the number of p73+ cells in the HFSR ( D , P = 0.741, Student t-test). ( E–J ) Properties of Gfap -GFP-expressing cells in the HFSR. In both control and AMD3100-treated mice, Gfap -GFP+ cells (arrows) in the HFSR do not express the granule cell marker Prox1 ( E and I ), although a few GFP-positive cells express the neural progenitor marker NeuroD (arrowheads in F and J ). However, the neural stem cell marker Sox2 ( G and K ) and the proliferation marker Ki67 ( H and L ) are expressed in the majority of Gfap- GFP-positive cells in the HFSR (arrowheads) in both groups. Results of quantitative analysis are shown in M. The number of Gfap -GFP+/Sox2+ cells and Gfap -GFP+/Ki67+ cells increased in the AMD3100-treated mice (* P < 0.05, Student t-test). We counted 10 sections from each of 3 (control) or 4 (AMD3100-treated) animals. DG, dentate gyrus; F, fimbria; V, ventricle. Scale bars = 50 µm in A1, B1 , and E – L ; 20 µm in A2, A3, B2 , and B3 .

Article Snippet: The primary antibodies used were as follows: anti-CXCR4 UMB2 (1:200, rabbit monoclonal, Abcam), anti-GFP (1:1,000, chick polyclonal, Abcam), anti-GM130 (1:500, mouse monoclonal, BD Transduction Laboratories), anti-γ-tubulin (1:200, mouse monoclonal, Sigma), anti-Ki67 (1:500, rabbit polyclonal, Novocastra), anti-LAMP1 (1:1000, rat monoclonal, Abcam), anti-NeuroD (1:200, goat polyclonal, Santa Cruz Biotechnology, Inc.), anti-Prox1 (1:1,000, goat polyclonal, R&D Systems), anti-p73 (1:200, rabbit polyclonal, Santa Cruz Biotechnology, Inc.).

Techniques: Injection, In Utero, Expressing, Marker

Journal: Development (Cambridge, England)

Article Title: Mechanisms underlying WNT-mediated priming of human embryonic stem cells

doi: 10.1242/dev.200335

Figure Lengend Snippet: The EOMES motif is enriched in WNT/ACT-enhanced ATAC peaks. (A) Schematic depicting the hypothesis to be tested that increased mesendodermal (ME) gene expression requires a SMAD2/3 co-activator (X) that is induced by WNT. (B) Heatmap of the ATAC-seq signals that are enhanced in the WNT/ACT condition in comparison with WNT or ACT alone. (C) Probability of finding these WNT/ACT-enhanced peaks near genes (start of the gene −20 kb to end of the gene +20 kb) that are either sensitive or insensitive to WNT priming. ***P<0.001. (D) The EOMES motif is the most enriched motif, followed by brachyury (BRA), which is found in the WNT/ACT-enhanced peaks near genes that are WNT primed.

Article Snippet: The following primary antibodies and dilutions were used: SOX2 (rabbit monoclonal, Cell Signaling Technology, 3579, 1:200), brachyury (goat polyclonal, R&D Systems, AF2085, 1:150), brachyury (rabbit monoclonal, R&D Systems, MAB20851, 1:400), EOMES (mouse monoclonal, R&D Systems, MAB6166, 1:400), GSC (goat polyclonal, R&D Systems, AF4086, 1:200).

Techniques: Gene Expression, Comparison

Journal: PLoS ONE

Article Title: Contribution of Corneal Neovascularization to Dendritic Cell Migration into the Central Area during Human Corneal Infection

doi: 10.1371/journal.pone.0109859

Figure Lengend Snippet: Activin A expression was determined using anti-activin A antibodies (goat polyclonal; 1∶50; R&D Systems; Minneapolis, MN, USA). A. Case 17; infected endophthalmitis. Activin A was expressed in the corneal epithelial and the subepithelial space with inflammatory cells. B. Case 19; bacterial endophthalmitis. Activin A was expressed in the endothelium of newly formed blood vessels, which suggests the presence of vessel formation in the central cornea.

Article Snippet: HCE cells were incubated with 1 µg/mL anti-activin A antibody (goat polyclonal; R&D Systems) for 12 h. The HCE cells were then stimulated with 5 µg/mL LPS, and supernatants (group E) were collected.

Techniques: Expressing, Infection

Journal: PLoS ONE

Article Title: Contribution of Corneal Neovascularization to Dendritic Cell Migration into the Central Area during Human Corneal Infection

doi: 10.1371/journal.pone.0109859

Figure Lengend Snippet: A. Levels of activin A in the supernatants from group A (HCE cells), B (HCE cells stimulated with 5 µg/mL LPS), C (HCE cells co-cultured with 10 5 /well, and D (HCE cells co-cultured with imDCs and stimulated with LPS for 1 hour, 3 hours, and 6 hours). The levels of activin A increased in a time-dependent manner in supernatants from group B. After 6 hours, they were higher in groups B and D than in group C ( p <0.01, Uni-ANOVA with a Bonferroni correction). B. Levels of IL-6 in the supernatants of the groups described in A. The levels of IL-6 increased in a time-dependent manner in groups B, C, and D. The levels of IL-6 increased after 6 hours in supernatants from groups B, C, and D, but were undetectable in those from group A. There were no significant differences in the levels of IL-6 among the supernatants from groups B, C, and D after 6 hours. C. Levels of TNF-α in supernatants from the groups described in A. TNF-α was not detectable in those from group A. TNF-α levels increased significantly only in group D, and the increase was time-dependent ( p <0.01, Uni-ANOVA with a Bonferroni correction).

Article Snippet: HCE cells were incubated with 1 µg/mL anti-activin A antibody (goat polyclonal; R&D Systems) for 12 h. The HCE cells were then stimulated with 5 µg/mL LPS, and supernatants (group E) were collected.

Techniques: Cell Culture

Journal: Disease Markers

Article Title: Site-Specific Secretome Map Evidences VSMC-Related Markers of Coronary Atherosclerosis Grade and Extent in the Hypercholesterolemic Swine

doi: 10.1155/2015/465242

Figure Lengend Snippet: Differential protein expression between HF RCA and CTRL cases.

Article Snippet: The following antibodies were used: CATD (C20), goat polyclonal (Santa Cruz Biotechnology, USA), dilution 1 : 300, and CH3L1 goat polyclonal (R&D Systems), dilution 1 : 500.

Techniques: Expressing, Binding Assay, Migration, Marker

Journal: Journal of Neuroinflammation

Article Title: CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species

doi: 10.1186/1742-2094-9-173

Figure Lengend Snippet: CD137L signaling activates microglia in vitro . Microglia cells were grown on plates that had been coated with PBS (grey bars) or 10 μg/mL of Fc control protein (white bars) or 10 μg/mL of CD137-Fc protein (black bars). ( A ) Attachment and morphological changes of primary microglia were documented by photography (40× magnification) 1 h after plating. Scale bar: 20 μm. ( B ) Attachment and morphological changes of BV-2 and N9 cells and primary microglia were documented by photography (63×) 24 h after plating. Cells exposed to immobilized CD137-Fc protein developed long spiky projections* and became amoeboid cells^ with shortened protrusions # . Scale bar: 20 μm. ( C ) The concentrations of cytokines in supernatants of primary microglia were determined by ELISA at indicated time points. Depicted are means ± standard deviations of triplicate measurements. ( D ) The phagocytic capacity of the cells was determined by adding FITC-labeled latex beads for 1 h before analysis by flow cytometry. Control: Autofluorescence of the cells. Numbers above the histograms state the percentages of positive cells and mean fluorescence intensities (MFI). These experiments were repeated three times with similar results. * P <0.05; ** P <0.01.

Article Snippet: Unspecific staining was blocked by 2% serum for 30 min. Endogenous peroxidases were inactivated by 3% hydrogen peroxide for 15 min. Anti-CD137 (goat polyclonal, R&D Systems) and anti-Iba-1 (rabbit polyclonal, Wako Chemicals) in PBS were used as primary antibodies and hybridized overnight.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species

doi: 10.1186/1742-2094-9-173

Figure Lengend Snippet: CD137L signaling activates microglia in vivo . ( A ) CD137L is required for activation of microglia in vivo . Cortex and spinal cord tissue sections of WT and CD137L -/- mice with EAE were immunohistochemically stained with an isotype control antibody or for Iba-1 (brown). Shown in the inset is a close-up of a single Iba-1-positive microglia cell in the cortex of a WT mouse with EAE. ( B ) Quantification of Iba-1 + microglia in the spinal cords and cortices of WT and CD137L -/- mice with EAE. Evaluated were three fields from two sections each using the Metamorph NX Software. Depicted are means ± standard deviations. ( C ) CD137 is expressed in the CNS during EAE. Spinal cord of naïve WT mice and WT mice with EAE was sectioned and stained with an isotype control antibody or for CD137 (brown). ( D ) The presence of CD137L is required for oligodendrocyte apoptosis in EAE. Tissue sections from the dorsal column of the spinal cord and the white matter of the cerebellum of WT and CD137L -/- mice with EAE were stained for oligodendrocytes using a Cy3-labeled anti-Nogo-A antibody (red). Apoptosis was detected by TUNEL staining (green). Nuclei were visualized by DAPI (blue). The yellow staining results from an overlay of red and green and indicates apoptotic oligodendrocytes. Magnification: 40×. ( E ) Quantification of apoptotic oligodendrocytes in the cerebellum of WT and CD137L -/- mice with EAE. Evaluated were three fields from two sections each using the Metamorph NX Software. Depicted are means ± standard deviations.

Article Snippet: Unspecific staining was blocked by 2% serum for 30 min. Endogenous peroxidases were inactivated by 3% hydrogen peroxide for 15 min. Anti-CD137 (goat polyclonal, R&D Systems) and anti-Iba-1 (rabbit polyclonal, Wako Chemicals) in PBS were used as primary antibodies and hybridized overnight.

Techniques: In Vivo, Activation Assay, Staining, Software, Labeling, TUNEL Assay

Journal: Journal of Neuroinflammation

Article Title: CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species

doi: 10.1186/1742-2094-9-173

Figure Lengend Snippet: Induction of oligodendrocyte death by CD137L-activated microglia. (A ) Expression of CD137L on the oligodendrocyte cell line OLN93 was determined by flow cytometry. Open histogram: Isotype control. Grey histogram: Anti-CD137L monoclonal antibody (clone TKS-1). ( B ) N9 and OLN93 cells were cultured for 24 h at a 1:1 ratio (1.5×10 5 each) on plates that had been coated with nothing (PBS) or 10 μg/mL of Fc control protein or 10 μg/mL of CD137-Fc protein. ( C ) The rates of OLN93 cell apoptosis in co-cultures with primary microglia from WT or CD137L -/- mice was determined 48 h after initiation of CD137L signaling by 7-AAD and Annexin V staining. Numbers in quadrants indicate percentages of cells. These experiments were repeated two to three times with similar results. * P <0.05; ** P <0.01.

Article Snippet: Unspecific staining was blocked by 2% serum for 30 min. Endogenous peroxidases were inactivated by 3% hydrogen peroxide for 15 min. Anti-CD137 (goat polyclonal, R&D Systems) and anti-Iba-1 (rabbit polyclonal, Wako Chemicals) in PBS were used as primary antibodies and hybridized overnight.

Techniques: Expressing, Flow Cytometry, Cell Culture, Staining

Journal: Journal of Neuroinflammation

Article Title: CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species

doi: 10.1186/1742-2094-9-173

Figure Lengend Snippet: CD137L-activated microglia induces oligodendrocyte apoptosis via ROS. ( A ) BV-2 cells were cultured on uncoated plates (PBS) or plates coated with Fc or CD137-Fc protein for 24 h, and were then stimulated with 0.4 μg of PMA for 1 h, stained with DHR123 before production of ROS was quantified by flow cytometry. White histogram: No DHR123. The number in the panel indicates the percentage of positive cells. ( B ) BV-2 cells were co-cultured with OLN93 cells at a 1:1 ratio with or without 10,000 U/mL catalase. The rate of apoptosis of cultures was determined after 24 h by 7-AAD and Annexin V staining. Numbers in quadrants indicate percentages of cells. ( C ) The percentages of 7-AAD + OLN93 cells with and without catalase treatment of B are presented as means ± standard deviations of triplicate measurements. * P < 0.05; ** P < 0.01.

Article Snippet: Unspecific staining was blocked by 2% serum for 30 min. Endogenous peroxidases were inactivated by 3% hydrogen peroxide for 15 min. Anti-CD137 (goat polyclonal, R&D Systems) and anti-Iba-1 (rabbit polyclonal, Wako Chemicals) in PBS were used as primary antibodies and hybridized overnight.

Techniques: Cell Culture, Staining, Flow Cytometry

Journal:

Article Title: Coronary and Aortic Endothelial Function Affected by Feedback between Adiponectin and TNF? in Type 2 Diabetic Mice

doi: 10.1161/ATVBAHA.110.214700

Figure Lengend Snippet: Role of adiponectin in improving coronary arteriolar endothelium-dependent and - independent vasodilation in type 2 diabetic mice. (A) and (B), ACh-induced vasodilation and flow-induced vasodilation (FID) were blunted in coronary arterioles of Leprdb, and adiponectin partially restored NO-mediated coronary arteriolar dilation to ACh and FID in Leprdb. (C) Endothelium-independent vasodilation of coronary arterioles to sodium nitroprusside (SNP) was not different among m Leprdb, Leprdb, and Leprdb+adiponectin. Data shown as mean±SEM. n=6 mice. *p<0.05 vs. m Leprdb; #p<0.05 vs. Leprdb.

Article Snippet: Primary antibodies for adiponectin (goat polyclonal, R&D, AF1119), and endothelial cell marker, von Willebrand factor (rabbit polyclonal, Abcam), or smooth muscle α-actin (rabbit polyclonal, Abcam) or fibroblast (rat monoclonal, Novus Biologicals) were used for sequential double immunofluorescence staining.

Techniques:

Journal:

Article Title: Coronary and Aortic Endothelial Function Affected by Feedback between Adiponectin and TNF? in Type 2 Diabetic Mice

doi: 10.1161/ATVBAHA.110.214700

Figure Lengend Snippet: Role of adiponectin and TNFα in aortic endothelium-dependent and - independent vasorelaxation in type 2 diabetic mice. (A) and (B), Endothelium-dependent vasorelaxation in response to ACh was significantly impaired in aortas of Leprdb. Adiponectin partially restored impaired vasorelaxation. Endothelium-independent vasorelaxation of aortic rings to SNP were similar among m Leprdb, Leprdb, and Leprdb+adiponectin. (C) and (D) Anti-TNFα (anti-TNF) improved endothelium-dependent vasorelaxation in Leprdb, but recombinant TNFα (TNF) treatment impaired ACh-induced vasorelaxation in m Leprdb. SNP-induced vasorelaxation of aortic rings were not different among m Leprdb, Leprdb, Leprdb+anti-TNF, and m Leprdb+TNF. Data represent mean±SEM. n=4-10 mice. *p<0.05 vs. m Leprdb; #p<0.05 vs. Leprdb.

Article Snippet: Primary antibodies for adiponectin (goat polyclonal, R&D, AF1119), and endothelial cell marker, von Willebrand factor (rabbit polyclonal, Abcam), or smooth muscle α-actin (rabbit polyclonal, Abcam) or fibroblast (rat monoclonal, Novus Biologicals) were used for sequential double immunofluorescence staining.

Techniques: Recombinant

Journal:

Article Title: Coronary and Aortic Endothelial Function Affected by Feedback between Adiponectin and TNF? in Type 2 Diabetic Mice

doi: 10.1161/ATVBAHA.110.214700

Figure Lengend Snippet: Reciprocal regulation between adiponectin and TNFα. (A) and (B), Protein expression of TNFα in isolated coronary arterioles and aortas was higher in Leprdb vs. m Leprdb, but adiponectin attenuated TNFα expression in Leprdb. (C) and (D), Adiponectin expression in coronary arterioles and aortas was reduced in Leprdb vs. m Leprdb, but anti-TNFα increased adiponectin expression in Leprdb. Data represent mean±SEM. n=4 separate experiments. *p<0.05 vs. m Leprdb; # p<0.05 vs. Leprdb.

Article Snippet: Primary antibodies for adiponectin (goat polyclonal, R&D, AF1119), and endothelial cell marker, von Willebrand factor (rabbit polyclonal, Abcam), or smooth muscle α-actin (rabbit polyclonal, Abcam) or fibroblast (rat monoclonal, Novus Biologicals) were used for sequential double immunofluorescence staining.

Techniques: Expressing, Isolation

Journal:

Article Title: Coronary and Aortic Endothelial Function Affected by Feedback between Adiponectin and TNF? in Type 2 Diabetic Mice

doi: 10.1161/ATVBAHA.110.214700

Figure Lengend Snippet: Co-localization and regulation of adiponectin expression in coronary microvessels. Dual fluorescence combining adiponectin with markers for endothelial cells [von Willebrand factor (vWF)], vascular smooth muscle (α-actin) and fibroblast marker with the use of specific primary antibodies followed by fluorescent-labeled secondary antibodies. A, B and C, dual labeling of adiponectin (red) and vWF (green) in control mouse heart tissue. D, E and F, dual labeling of adiponectin (red) and vWF (green) in Leprdb mouse heart tissue. G, H and I, dual labeling of adiponectin (red) and vWF (green) in dbTNF-/dbTNF- heart tissue. Blue arrows in C, F and I show the colocalization of adiponectin and endothelial cells (yellow). J, K and L, dual labeling of adiponectin (red) and α-actin (green) in control mouse heart tissue. The pink arrow in L shows the specific α-actin staining with absence of adiponectin staining. M, N and O, dual labeling of adiponectin (red) and marker of fibroblast in Leprdb mice heart tissue. The brown arrow in O shows the specific fibroblast staining with absence of adiponectin staining. P and Q, negative control: the purple arrows show an absence of staining in vessels with isotype control IgG and without primary antibodies. R shows nuclear staining with DAPI (blue) in control mice heart tissue. Magnification×40. Data shown are representative of 4 separate experiments.

Article Snippet: Primary antibodies for adiponectin (goat polyclonal, R&D, AF1119), and endothelial cell marker, von Willebrand factor (rabbit polyclonal, Abcam), or smooth muscle α-actin (rabbit polyclonal, Abcam) or fibroblast (rat monoclonal, Novus Biologicals) were used for sequential double immunofluorescence staining.

Techniques: Expressing, Fluorescence, Marker, Labeling, Staining, Negative Control

Journal:

Article Title: Coronary and Aortic Endothelial Function Affected by Feedback between Adiponectin and TNF? in Type 2 Diabetic Mice

doi: 10.1161/ATVBAHA.110.214700

Figure Lengend Snippet: Protein expression of adiponectin receptors in coronary arterioles and aortas. (A) and (B), Adiponectin receptor 1 (AdipoR1) protein expression was not significantly different between m Leprdb and Leprdb in both coronary arterioles and aortas. (C) and (D), adiponectin receptor 2 (AdipoR2) protein expression was remarkably decreased in Leprdb coronary arterioles and aortas. Data represent mean±SEM. n=4 separate experiments. *p<0.05 vs. m Leprdb; #p<0.05 vs. Leprdb.

Article Snippet: Primary antibodies for adiponectin (goat polyclonal, R&D, AF1119), and endothelial cell marker, von Willebrand factor (rabbit polyclonal, Abcam), or smooth muscle α-actin (rabbit polyclonal, Abcam) or fibroblast (rat monoclonal, Novus Biologicals) were used for sequential double immunofluorescence staining.

Techniques: Expressing

Journal:

Article Title: Coronary and Aortic Endothelial Function Affected by Feedback between Adiponectin and TNF? in Type 2 Diabetic Mice

doi: 10.1161/ATVBAHA.110.214700

Figure Lengend Snippet: Adiponectin and anti-TNFα inhibit IκBα phosphorylation and NFκB expression. In both coronary arterioles and aortas, total inhibitor of NFκB (IκBα) expression was decreased in Leprdb (A and D) while phospho-IκBα was greatly elevated (B and E). Both adiponectin and anti-TNFα inhibited IκBα phosphorylation (B and E) without affecting the total IκBα expression (A and D). (C) and (F), Nuclear factor-kappa B (NFκB) p65 protein expression was significantly increased in Leprdb. Adiponectin and anti-TNFα decreased NFκB protein expression. Data represent mean±SEM. n=4 separate experiments. *P<0.05 vs. m Leprdb; #P<0.05 vs. Leprdb.

Article Snippet: Primary antibodies for adiponectin (goat polyclonal, R&D, AF1119), and endothelial cell marker, von Willebrand factor (rabbit polyclonal, Abcam), or smooth muscle α-actin (rabbit polyclonal, Abcam) or fibroblast (rat monoclonal, Novus Biologicals) were used for sequential double immunofluorescence staining.

Techniques: Expressing