The following antibodies and chemicals were used for this study: rabbit anti-α-tubulin (Abcam catalog #ab15246, RRID: AB_301787 ; 1:500), Rabbit anti-β3-tubulin (TUBB3; Covance catalog #MMS-435P, RRID: AB_2313773 , 1:2000), mouse anti-MBP (BioLegend catalog #836504, RRID: AB_2616694 ; 1:1000), goat anti-Olig2 (R&D Systems catalog #AF2418, RRID: AB_2157554 ; 1:20), and mouse anti-O4 IgM (R&D Systems catalog #MAB 1326, RRID: AB_357617 ; 1:200). Alexa Fluor-conjugated secondary antibodies were all purchased from Thermo Fisher Scientific with a dilution of 1:500. nifedipine, verapamil, diltiazem, NNC 55-0396, ω-conotoxin GVIA (ω-CTX), ω-agatoxin IVA, SKF96365, cyclopiazonic acid (CPA), thapsigargin (Tg), and ryanodine (Ry) were all purchased from Sigma.
Af2418
Manufactured by R&D Systems
Sourced in United States
AF2418 is a recombinant human Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) protein. GM-CSF is a cytokine that plays a role in the differentiation and activation of granulocytes and macrophages.
Automatically generated - may contain errors
Lab products found in correlation
Mab1326, by R&D Systems (3 mentions)
Ab126783, by BD (2 mentions)
Dm4000b microscope, by Leica camera (2 mentions)
Anti h2k119ub, by Cell Signaling Technology (2 mentions)
Scn400 slide scanner, by Leica camera (2 mentions)
Anti h3k27me3, by Merck Group (2 mentions)
Lactacystin, by Merck Group (2 mentions)
Ab126783, by Cell Signaling Technology (2 mentions)
Ab86428, by BD (2 mentions)
Ab89260, by Santa Cruz Biotechnology (2 mentions)
α tubulin, by Merck Group (2 mentions)
Sc 9133, by Merck Group (2 mentions)
Pierce crosslink magnetic ip and co ip kit, by Thermo Fisher Scientific (2 mentions)
Af2018, by Abcam (2 mentions)
Ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (2 mentions)
17 protocols using af2418
1
Immunoprofiling of Oligodendrocyte Lineage
2
Glioma Molecular Profiling via TMA
3
Characterization of BMI1 Ubiquitination
4
Glioma Molecular Profiling via TMA
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5 μm thick slides of de-identified paraffin-embedded tissue microarrays (TMAs) were constructed from glioma after obtaining Ohio State University Institutional Review Board Approval. A total of 96 cases were arrayed on the TMA block, including 15 non-neoplastic controls (cortical dysplasias), 16 grade II glioma cases, 27 grade III gliomas, and 38 grade IV glioblastomas. Tissues too small and/or crushed on the TMA were eliminated from analysis after immunohistochemistry staining with anti-BMI1 (1:50, Abcam, ab126783), anti-EZH2 (1:100, BD Biosciences, 612667), anti-CD44 (1:100, BD Biosciences, 550392), anti-OLIG2 (1:100, R&D system, AF2418), and secondary HRP-conjugated antibodies. The TMA images were taken by a Leica SCN400 Slide Scanner. Overall staining on TMA was scored as – (negative) or + (positive) compared to non-neoplastic controls. PN3691 (with or without treatment with BMI1 or EZH2 inhibitors) xenografted brain tissues were fixed in 4% paraformaldehyde, and were stained with anti-H2K119Ub (1:100, Cell signaling, #8240S), anti-H3K27Me3 (1:1000, Millipore, 07-689), and secondary HRP-conjugated antibodies. Nuclei in immunohistochemistry were counterstained with Hematoxylin. The immunohistochemistry images were taken by a LEICA DM4000B Microscope, and were analyzed by IHC Profiler.
5
Characterization of BMI1 Ubiquitination
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Cells were collected and lysed in IP Lysis buffer (Thermo Scientific) containing phosSTOP phosphatase inhibitor cocktail (Roche) and protease inhibitor cocktail (Sigma, St. Louis) and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (NuPAGE Bis-Tris gel, Invitrogen) and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% (wt/vol) non-fat milk in PBS-Tween-20 (0.5% vol/vol) and probed with primary antibodies against BMI1 (1:1000, Abcam, ab126783), H2K119Ub (1:1000, Cell signaling, #8240S), CD44 (1:1000, BD Biosciences, 550392), YKL40 (1:1000, Abcam, ab86428), EZH2 (1:1000, BD Biosciences, 612667), H3K27Me3 (1:1000, Millipore, 07-689), OLIG2 (1:1000, R&D system, AF2418), SOX2 (1:1000, R&D system, AF2018), RNF144A (1:500, Abcam, ab89260), Ub (1:5000, Santa Cruz, sc-9133), TUBULIN (α-Tubulin,1:10,000, Sigma-Aldrich, T6074), and ACTIN (β-actin,1:10,000, Sigma-Aldrich, A1978). BMI1-RNF144A interaction and BMI1 polyubiquitination were detected by Pierce Crosslink magnetic IP and Co-IP kit (Thermo Scientific). For the ubiquitination assays, cells were treated with Lactacystin (5 μM or 10 μM; Sigma) for 5hr before collection. BMI1 polyubiquitination was quantified by Image J.
6
Immunostaining Markers for Cellular Analysis
7
Immunocytochemistry of Neural Cell Types
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Cells were fixed in 4% paraformaldehyde (PFA; Sigma, St Louis, MO, USA) at room temperature for 15 min, rinsed with phosphate-buffered saline (PBS), and permeabilized with 0.2% Triton X-100 containing 5% normal donkey serum for 30 min at room temperature. Cells were then labeled with primary antibodies for 2 h at room temperature or overnight at 4 °C with the following conditions: HB9 (1:80, 81.5C10, DSHB), CHAT (1:200, AB144P, Millipore-Merck), Olig2 (1:80, AF2418, R&D), β3-Tubulin (1:1000, T8578, Sigma or MRB-435P-100, Biolegend), Vimentin (1:100, M7020, Dako), S100b (1:200, S2532, Sigma), GFAP (1:200, Z0334, Dako), O4 (1:150, MAB-1326, R&D), NeuN (1:400, D3S3I, Cell Signaling Technology). All antibodies were diluted in PBS containing 5% serum and 0.2% Triton X-100. The cells were washed the following day and incubated with Cy3-, Cy2- and Cy5-conjugated antibodies (Jackson Immunoresearch Laboratories) for double-staining experiments and processed for visualization using standard protocols.
8
Immunocytochemistry Profiling of Neuronal Markers
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Immunocytochemistry was performed using standard protocols. Cells were incubated overnight at 4 °C with primary antibodies. The following proteins were evaluated: Tbr1 (ab31940, Abcam, 1:500), HB9 (81.5C10, DSHB, 1:80), CHAT (AB144P, Millipore-Merck, 1:200), Olig2, β3-Tubulin (Sigma T8578, 1:1000), (AF2418, R&D, 1:80), Anti-NRP1 (GTI5036, Neuromics, 1:200). All antibodies were diluted in PBS containing 5% serum and 0.1% Triton X-100. The cells were washed the following day and incubated with Cy3-, Cy2-, and Cy5-conjugated antibodies (Jackson Immunoresearch Laboratories) for double-staining experiments, and processed for visualization using standard protocols.
9
Immunofluorescence Labeling of Oligodendrocytes
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Immunofluorescence double labeling was performed as previously described.13 The primary antibodies (Ab) were goat anti‐Olig2 (1:200 dilution, AF2418, R&D Systems), rabbit anti‐Olig2 (1:200 dilution, ab109186, Abcam), rabbit anti‐Nogo‐A (1:100 dilution, ab62024, Abcam), and goat anti‐LCN2 (1:100, AF1757, R&D Systems). The secondary Abs were Alexa Fluor 488‐conjugated donkey anti‐rabbit mAb (1:500 dilution, Invitrogen), Alexa Fluor 488‐conjugated donkey anti‐goat mAb (1:500 dilution, Invitrogen), Alexa Fluor 594‐conjugated donkey anti‐rabbit mAb (1:500 dilution, Invitrogen), and Alexa Fluor 594‐conjugated donkey anti‐goat mAb (1:500 dilution, Invitrogen). Negative controls omitted the primary antibodies.
Olig2 is a helix–loop–helix transcription factor found in all OPCs, immature oligodendrocytes, and mature oligodendrocytes. Thus, Olig2+ cell numbers reflect all oligodendrocytes. In contrast, Nogo‐A identifies only the mature phenotype. Cells which were Nogo‐A−/Olig2+ cells were subclassified as oligodendrocyte precursor cells/immature oligodendrocytes.16 , 26 , 27
Olig2 is a helix–loop–helix transcription factor found in all OPCs, immature oligodendrocytes, and mature oligodendrocytes. Thus, Olig2+ cell numbers reflect all oligodendrocytes. In contrast, Nogo‐A identifies only the mature phenotype. Cells which were Nogo‐A−/Olig2+ cells were subclassified as oligodendrocyte precursor cells/immature oligodendrocytes.
10
Immunofluorescent Staining of Brain Sections
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Brain and spinal cord sections from the SVZ and LE in the mice of the four groups were deparaffinized, boiled with citrate buffer at 95°C for 20 min, cooled to 30°C, and blocked with 1% bovine serum albumin (BSA) at 37°C for 1 h. The sections were then incubated overnight at 4°C with primary antibodies against Olig2 (1 : 400; AF2418; R&D Systems, Minneapolis, USA), CC-1 (1 : 200; OP80; Merck Chemicals, Darmstadt, Germany), iNOS (1 : 200; ab210823; Abcam, Cambridge, UK), Arginase 1 (Arg1; 1 : 200; ab91279; Abcam, Cambridge, UK), Iba1 (1 : 200; ab48004; Abcam, Cambridge, UK), followed by 1 h of incubation with FITC- (1 : 400-) and TRITC- (1 : 200-) conjugated secondary antibodies (Southern Biotech, Birmingham, USA) at 37°C. Nuclei were counterstained with DAPI (Southern Biotech, Birmingham, USA). We used a fluorescence microscope to capture the images and then analyzed them using ImageJ.
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