Paraffin‐embedded coronal sections of brains were stained with Luxol fast blue (LFB, Sigma, S3382) to assess remyelination. Images were taken and quantitative image analysis was performed using Image‐Pro Plus. Region of corpus callosum was initially marked using the “irregular AOI” tool, blue areas were then counted within the lesion using the “count and measure objects” tool. Percentage of the remyelination area was calculated by the ratio of the blue area and total corpus callosum area. For immunofluorescent analysis, frozen sections of brains and spinal cords were blocked and permeated with PBS containing 2.5% BSA and 0.3% Triton‐X 100 for 45 min at room temperature, then incubated with mouse anti‐MBP antibody (Covance, SMI‐94R, 1:500), mouse polyclonal anti‐MOG antibody (Millipore, AB5320, 1:500) and rabbit polyclonal anti‐GST‐pi antibody (Millipore, AB5320, 1:500), rabbit anti‐PDGFRα (Cell signaling, 3164S, 1:200), and rabbit anti‐NG2 (Millipore, AB5320, 1:200) at 4°C overnight. After thorough washing, the sections were stained with secondary antibody conjugated to Alexa Fluor 488 or Alexa Fluor 555 (Thermo Fisher, 1:1,000) for 1 hr at room temperature, and nuclei were stained with Hoechst 33342. Images were taken using an Olympus IX71 inverted fluorescent microscope, and quantitative image analysis was performed using Image‐Pro Plus.
Ab5320
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
The AB5320 is a piece of laboratory equipment manufactured by Merck Group. It is used for the measurement and analysis of various samples. The core function of the AB5320 is to provide accurate and consistent data for research and testing purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ab9610, by Merck Group (6 mentions)
Mab386, by Merck Group (5 mentions)
Lsm 700, by Zeiss (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Ab39012, by Abcam (4 mentions)
Lsm 710, by Zeiss (4 mentions)
Ab6326, by Abcam (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Mabn50, by Merck Group (3 mentions)
Mab360, by Merck Group (3 mentions)
Z0334, by Agilent Technologies (2 mentions)
A21434, by Thermo Fisher Scientific (2 mentions)
Ab32570, by Abcam (2 mentions)
Ab75769, by Abcam (2 mentions)
Ab24590, by Abcam (2 mentions)
A11012, by Thermo Fisher Scientific (2 mentions)
Lsm 780, by Zeiss (2 mentions)
C8035, by Merck Group (2 mentions)
Mab377, by Merck Group (2 mentions)
Mab1637, by Merck Group (2 mentions)
71 protocols using ab5320
1
Quantitative Analysis of Myelination in Neurological Conditions
2
Immunofluorescence Analysis of Tumor Vasculature
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Tumor tissue samples or healthy tissues from non-tumor-bearing mice were collected and fixed overnight with 4% PFA. Tissue samples were cut into thin pieces and digested for 5 min with 20 mM proteinase K in a 10 mM Tris-buffer (pH 7.5), followed by incubation with 100% methanol for 30 min. Samples were incubated overnight at 4 °C in 0.3% Triton X-100 PBS containing 3% skim milk. After washing several times with PBS, tissue samples were incubated with a combination of a rat anti-mouse CD31 (1:200; 553370; BD Pharmingen) antibody and a rabbit polyclonal anti-NG2 (1:200; AB5320; Merck) antibody, followed by incubation with secondary antibodies, consisting of an Alexa Fluor 555-labeled goat anti-rat (1:200; A21434; Thermo Fisher SCIENTIFIC) and an Alexa Fluor 647-labeled goat anti-rabbit (1:200; A-21244; Thermo Fisher SCIENTIFIC). After washing with PBS, tissues were mounted using a vectashield-mounting medium (H1000; Vector Laboratories) and images were taken by confocal microscopy (Nikon C1 Confocal microscope, Nikon Corporation, Japan). Three-dimensional images of tumor vessels were analyzed. Positive signals of CD31 or NG2 area were calculated using an Adobe Photoshop software (CS6; Adobe) program. Pericyte coverage was quantified as a percentage of vessel areas covered by pericytes with a calculation of overlap area of CD31 and NG2 positive signals.
3
Immunofluorescence Analysis of Vascularized Organoids
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For immunofluorescence analyses, vascularized organoids were fixed in 4% PFA solution for 30 min, washed in PBS and embedded into an agarose gel (1%). Afterward, 5 µm paraffin sections were prepared. Sections were deparaffinized, rehydrated and stained with eosin and hematoxylin. For immunofluorescence analyses, antigens were unmasked using Sodium Citrate buffer (10 mM, pH6). Primary antibodies to TUJ1 (Biozol, 801202), GFAP (DAKO, Z0334), CD31 (DAKO, M0823), Iba1 (WAKO, 019-19741), Sox1 (R&D Systems, AF3369), Pax6 (Biolegend, 901301), Brachyury (T) (R&D Systems, AF2085), MAP2 (Abcam, AB32454), N-Cadherin (Sigma-Aldrich, C3865) and NG2 (Merck-Millipore, AB5320) were used. Sections were incubated with primary antibodies overnight at 4 °C. Secondary Cy2‐, Cy3- or Cy5-labelled antibodies were used to visualize primary antibodies. Sections were incubated with secondary antibodies for 2 h at room temperature. All antibodies were diluted in blocking solution.
4
Immunochemical Identification of CNS Cell Types
5
Endothelial Cell Identification in Cocultures
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To demonstrate the presence of endothelial cells in the cocultures, CD31 immunofluorescence staining was conducted on half a sample fixed on day 10. After permeabilization, nonspecific protein binding was blocked before 1 hr of incubation with the primary anti‐CD31 antibody (0.13 mg/ml mouse anti‐human CD31, M0823, Dako), with or without anti‐NG2 chondroitin sulfate proteoglycan antibody (0.001 mg/ml, Merck Millipore, AB5320). After washing with 0.1% Tween in PBS, 1 hr of incubation with the secondary antibody (1:200, sheep anti‐mouse biotinylated, RPN1001v1, GE Healthcare) was performed. The samples were incubated with streptavidin Alexa Fluor 488, 2 mg/ml (Invitrogen), together with the anti‐α‐smooth muscle actin (α‐SMA) fluorescently labelled antibody (Clone 1A4, Cy3 0.5 μg/ml, C6198, Sigma Aldrich). The 5 mm Ø × 5 mm constructs were incubated overnight (4°C) with the antibodies. Finally, nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI) staining 100 ng/ml (Sigma) for 15 min at room temperature.
6
Immunofluorescence Quantification of Oligodendrocyte Lineage Cells
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Dissociated PAR1+/+ or PAR1−/− NSCs at passage 3 were plated as monolayers in triplicate on poly-L-lysine coated glass cover slips and grown for 2 DIV in media containing EGF and bFGF or for 5 DIV after growth factor withdrawal. At each end point, cultures were fixed with 2% paraformaldehyde and stained for NG2 (AB5320, EMD Millipore), Olig2 (AB9610, EMD Millipore), or PLP (ab28486, Abcam) using immunofluorescence techniques. Species appropriate fluorochrome conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Sections were cover slipped with VECTASHIELD Hardset containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA). Five 20X microscopic fields encompassing the center and 4 poles of each coverslip were digitally imaged using an Olympus BX51 microscope (Olympus, Center Valley, PA). The mean number of NG2+ or Olig2+ cells was enumerated without knowledge of genotype and expressed as a ratio of the total number of DAPI+ cells counted in the same fields.
7
Immunohistochemical Analysis of Histone Modifications
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Mice were anesthetized and then perfused, cryopreserved, embedded, and sectioned as previously described (Liu et al., 2012 (link)). Immunohistochemistry was performed as previously described (Liu et al., 2012 (link)) with primary antibodies against trimethylated histone 3 lysine 9 (H3K9me3, 1:100; ab8898, Abcam), CC1 (1:100; OP80, Calbiochem), myelin basic protein (MBP, 1:500; SMI99, Covance), OLIG2 (1:200, ab81093, Abcam), NG2 (1:200; AB5320, EMD Millipore) or Caspr (1:100, ab34151, Abcam). Stained sections were visualized using confocal microscopy (LSM800 Meta confocal laser scanning microscope, Carl Zeiss Micro-Imaging). For NG2, CC1, OLIG2 cell counts, and H3K9me3 intensity quantifications, 4–6 20x fields were taken per mouse. For MBP-covered segments and internodal length marked by Caspr, 4–6 fields were taken per mouse followed by quantifications using ImageJ. One-way ANOVA tests were performed to assess statistical differences. For correlation of internodal length with social interaction ratio, data normality was determined using D’Agostino and Person test in GraphPad Prism 8. Pearson correlation coefficients were calculated if data passed normality test.
8
Pericyte Isolation and Characterization
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Pericytes were isolated and cultured as previously described [23 (link)]. Morphological characterization was performed using phase contrast microscopy, and immunofluorescence characterization was performed using confocal laser scanning microscopy (CLSM; SP5II, Leica Microsystems, Wetzlar, Germany). The cells were verified with primary antibodies against PDGFR-β (ab32570, Abcam, Cambridge, UK), nerve/glial antigen 2 (NG-2; ab5320, Merck Millipore, Burlington, MA, USA), CD146 (ab75769, Abcam), α-smooth muscle actin (α-SMA; ab7817, Abcam), and platelet endothelial cell adhesion molecule (CD31; ab24590, Abcam).
For flow cytometry, cells were labeled with directly conjugated antibodies, including NG-2-PE, CD146-PE, PDGFR-β-PE, CD31-PE and IgG-PE (all from BD Biosciences, Franklin Lakes, NJ, USA). Samples were analyzed using the high-sensitivity imaging flow cytometer Amnis ImageStream MK II (ISX).
For flow cytometry, cells were labeled with directly conjugated antibodies, including NG-2-PE, CD146-PE, PDGFR-β-PE, CD31-PE and IgG-PE (all from BD Biosciences, Franklin Lakes, NJ, USA). Samples were analyzed using the high-sensitivity imaging flow cytometer Amnis ImageStream MK II (ISX).
9
Retinal Vascular Pericyte Analysis
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The eyes were rapidly enucleated and fixed in 4% paraformaldehyde (PFA) for 2 min before being dissected in flower shape in 2× phosphate-buffered saline (PBS). The retinae were fixed again in 100% methanol and stained with primary antibodies against CD31 (rat monoclonal, 553370, BD Biosciences, San Jose, CA, USA, RRID:AB_394816) and NG2 (rabbit polyclonal, AB5320, Merck Millipore, Germany, RRID:AB_11213678) overnight at 4 °C, followed by incubation with Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (A-11006 or A-11012, Thermo Fisher Scientific, USA) and 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, USA). The retinae were flatmounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific, USA) and examined by confocal microscopy (Zeiss LSM 800, Zeiss, Germany). The retinal vasculature was analyzed using AngioTool [62 (link)]. Pericyte coverage was determined by the ratio of NG2+ area/CD31+ area.
10
Immunohistochemical Analysis of TMEV Infection
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Immunohistochemistry was performed on fixed tissue from six animals from each experimental group. The mice were anesthetized with pentobarbital (50 mg/kg body weight, i.p.) and perfused with saline on day 35 and 60 post-TMEV infection (p.i), and the brain of the mice was fixed overnight in 4% PFA prepared in 0.1 M phosphate buffer saline (PBS). Free-floating coronal vibratome sections of the brain (30 μm thick) were washed three times in PBS, incubated with PBS containing 0.1% Triton-X100 (PBT), and blocked for 1 h at room temperature in blocking buffer: PBT containing 5% normal serum (Vector Laboratories). Sections were then incubated overnight at 4 °C with primary antibodies against the following proteins: β-Catenin (1:400, 04-958, Merck), CC1 (1:200, OP80, Calbiochem), Doublecortin (DCX–1:500, SC-8066, Santa Cruz Biotechnologies), GFAP (1:1000, G9269, Merck), Iba-1 (1:1000, 019-19741, Wako), NG2 (1:500, AB5320, Merck), Olig2 (1:200, sc-19967, Santa Cruz Biotechnologies), and γ-Tubulin (1:200, sc-7396, Santa Cruz Biotechnologies). On the following day, the sections were rinsed three times with PBT and they were then incubated for 1 h with an Alexa Fluor-conjugated (1:500, Molecular Probes Inc.) secondary antibody diluted in blocking buffer. In all cases, the specificity of staining was confirmed by omitting the primary antibody.
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