Brain single-cell suspensions from 9-mo-old WT mice were stained with Ghost Dye violet 510 (1:1,000; Tonbo Biosciences) and anti–CD16/CD32 antibody (1:100; BD Biosciences) followed by anti-CD11b (1:100; BioLegend), anti-CD45 (1:100; BioLegend), anti-CD11c (1:50; BioLegend), anti-TMEM119 (1:200; Abcam), and anti-CCR2 (1:100; BioLegend). CD11b+ cells were gated from single/live cells followed by subsequent gating of CD11b+CD45low as microglia and CD11b+CD45high as macrophages. To distinguish microglia and macrophages, the CCR2 marker expressed by blood-derived macrophages but not by microglia was used (3 (link), 16 (link)). To further distinguish these two cell types, the microglia-specific marker Tmem119 (17 (link)) was also included. The CD11b+CD45high cells that express CCR2 but not Tmem119 were confirmed as macrophages, while the CD11b+CD45low cells that all express Tmem119 but do not express CCR2 were confirmed as microglia. FMO negative controls were included to confirm the specificity of CD11c staining in CD11b+CD45low microglial populations. Brain CD45− cells that mainly contain nonimmune cells (e.g., neurons, astrocytes, and oligodendrocytes) that do not express CD11c were also included as negative controls to further validate the specificity of this strategy.
Anti tmem119
Manufactured by Abcam
Sourced in United States
Anti-TMEM119 is a protein marker that recognizes the transmembrane protein TMEM119, which is expressed on the surface of microglia cells in the central nervous system. This antibody can be used to identify and study microglia in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti iba1, by Fujifilm (3 mentions)
Anti cd68, by Bio-Rad (3 mentions)
Anti iba1, by Abcam (3 mentions)
Axiovert 200m, by Zeiss (2 mentions)
Las af, by Leica (2 mentions)
Efficient navigation zen , by Zeiss (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Anti map2ab, by Merck Group (2 mentions)
Anti βiii tubulin, by Fortrea (2 mentions)
Anti ki67, by Abcam (2 mentions)
Anti inos, by Abcam (2 mentions)
Anti cd16 cd32 antibody, by BD (1 mentions)
Anti cd45, by BioLegend (1 mentions)
Ghost dye violet 510, by Cytek Biosciences (1 mentions)
Anti ccr2, by BioLegend (1 mentions)
Anti cd11c, by BioLegend (1 mentions)
Anti cd11b, by BioLegend (1 mentions)
Axio scan z1 slide scanner, by Zeiss (1 mentions)
Aperio scanscope, by Leica (1 mentions)
Zen blue, by Zeiss (1 mentions)
11 protocols using anti tmem119
1
Distinguishing Microglia and Macrophages
2
Quantifying Neuroinflammation and Tau Pathology in DLFC
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Histological staining and analysis of total AT8 pTau density, Iba1+, and CD68+ cell count in the DLFC at the depth of the cortical sulcus was performed using the Aperio ScanScope (Leica) as previously described [26 (link)]. For immunofluorescence staining, tissue was extracted from the DLFC, embedded in paraffin, and cut at 20 μm. Immunofluorescence staining was performed using Akoya Bioscience Opal Polaris 7 color manual IHC detection kit as per the manufactures instructions as previously described (tau isoform paper when published). Sections were incubated with antibodies to anti-Iba1 (1:500, Wako), anti-PHF-tau (AT8) (1:1000, Pierce Endogen), anti-TMEM119 (1:100, Abcam), and DAPI. Stained sections were digitized using an Axio Scan.Z1 slide scanner (Zeiss) and visualized using Zen Blue (Zeiss).
3
Immunocytochemical Analysis of Neuronal and Glial Markers
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Cells were fixed in 4% paraformaldehyde for 10 min and blocked for 1 h at room temperature (RT) with blocking buffer (5% FBS and 0.3% Triton X-100). After blocking, the cells were incubated with the following primary antibodies for 2 h at RT: anti-βIII-tubulin (1:500, Covance), anti-Map2ab (1:500, Sigma), anti-GFP (1:500, Aves), anti-NeuroD1 (1:200, Abcam), anti-Tmem119 (1:500, Abcam), anti-CD68 (1:500, Bio-Rad), and anti-Iba1 (1:500, Abcam). Stained cells were visualized with a fluorescence microscope (Axiovert 200 M, Zeiss) and a confocal microscope (LSM800, Zeiss). Fluorescence intensity of the cell soma was quantified using LAS AF (Leica) or ZEN (Zeiss).
4
Immunostaining Markers for Tissue Analysis
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Antibodies for immunofluorescence (IF) and immunohistochemistry (IHC) were obtained and used as described in the following paragraph. Anti-GFAP (Z0334, rabbit, 1:1,000 for IHC) from DAKO, Agilent Technologies (Carpinteria, CA), anti-CD31 (77699, rabbit, 1:100 for IHC) from CST, Cell Signaling Technology, anti-Ki67 (ab15580, rabbit, 1:200 for IHC) from Abcam, anti-Iba1 (019-19741 rabbit, 1: 200 for IHC and 1:250 for IF) from Wako Chemicals United States, anti-Olig2 (EMD rabbit, 1:200 for IHC) from EMD Millipore. Anti-CD8 (ab209775, rabbit, 1:200 for IF) from Abcam, anti-GrB (AF 1865, goat, 1:100 for IF) from R&D Systems, anti-Tmem119 (ab209064, rabbit, 1:200 for IF) from Abcam, and anti-F4/80 (30325T, rabbit, 1:400 for IF) from Cell Signaling Technology.
5
Immunocytochemistry Staining of Microglia
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Cells were washed three times with DPBS (1X) and fixed with cold PFA (4% w/v) for 20 min at room temperature (RT) followed by three washes with PBS (1X). Cells were blocked with blocking solution (PBS with 0.1% Triton X-100 and 1% BSA) for 30 min at RT. Primary antibodies were added at respective dilutions (see below) in blocking solution and placed at 4°C overnight. The next day, cells were washed 3 times with PBS for 5 min and stained with Alexa Fluor conjugated secondary antibodies from Invitrogen (1:800) for 2.5 h at room temperature in the dark. After secondary staining, cells were washed 3 times with PBS and cover slipped with ProLong™ Diamond Antifade Mountant or Fluoromount-G™ (ThermoFisher). Primary antibodies used for ICC: anti-P2ry12 (1:500, HPA014518 Sigma), anti-TREM2 (1:200, AF1828 R&D Systems), anti-CD68 (1:100, M0718 Dako), anti-TMEM119 (1:100, ab185333 Abcam), anti-IBA1 (1:500; Wako, 019-19741).
6
Immunohistochemical and Flow Cytometric Analysis of Brain Markers
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For monocyte/macrophages expression anti-Ly6C (1:200 Abcam) was used and followed by anti-rat Alexa 488 (1:1000, Invitrogen). To visualise microglia/macrophages, resident microglia, claudin-5 and ZO-1, anti-Iba1 (1:1000 WAKO), anti-TMEM119 (1:500, Abcam), anti-claudin-5 (1:500, Abcam) and anti-zo-1 (1:500, ThermoFisher scientific) were used, respectively. All were followed by anti-rabbit Alexa 488 (1:1000, Invitrogen). To visualise CatS, anti-CatS (1:100, Santa Cruz) was used followed by anti-mouse Alexa 488 (1:1000, Invitrogen). For FACS analysis reported below the following antibodies were used: occludin monoclonal antibody OC-3F10 (1:100, Invitrogen) P-glycoprotein monoclonal antibody C213 (1:10, Thermo Fisher Scientific). ICAM-1/VCAM-1, anti-mouse CD54 (1:200, e-BioLegend) and anti-mouse CD106 (1:200, e-BioLegend). Secondary coupled antibody used for occludin and p-Glycoprotein was an anti-mouse Alexa 488 (1:200, Life Technology).
7
Immunohistochemistry Analysis Reagents
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Anti-Aβ clone 4G8 was purchased from Biolegend (San Diego, CA). Anti-α-tubulin antibody and the horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All mouse cytokine ELISA kits were obtained from R&D Systems (Minneapolis, MN). Elite Vectastain ABC avidin and biotin kits, biotinylated anti-rabbit, anti-mouse, and anti-rat antibodies and the Vector VIP kits were from Vector Laboratories Inc. (Burlingame, CA). Anti-CD68 was obtained from Serotec (Raleigh, NC). Anti-PSD95 and anti-GFAP antibodies were purchased from Cell Signaling Technology (Danvers, MA). Synaptophysin antibody was purchased from Chemicon International, Inc. (Temecula, CA) while anti-Cox-2, anti-TMEM119 and biotinylated anti-mouse IgG2b antibodies were from Abcam (Cambridge, MA). The Anti-Iba-1 antibody was from Wako Chemicals USA, Inc. (Richmond, VA). The Anti-CD4 antibody was purchased from BD Pharmingen (BD Biosciences, San Jose, CA).
8
Multimodal Nanoparticle-based Imaging Assay
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Bis(2,4,5-trichloro-6-carbopentoxyphenyl) oxalate (CPPO), Ficoll, DCFH-DAm and lipopolysaccharides (LPS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Alexa FlourTM-488-phalloidin and TAMRA were purchased from Thermo Fisher Scientific (Massachusetts, USA). DiR, DiO, DiD, propidium iodide, fluorescein isothiocyanate (FITC), 6-diamidino-2-phinylindolo dihydrochloride (DAPI)m and Cyanine 7 (Cy7) were obtained from Fanbo Biochemical (Beijing, China). AQ4N was purchased from MedChemExpress. Anti-CD9, anti-CD81, anti-ALIX, anti-TSG101, anti-Ki67, anti-iNOS, anti-CD163, anti-TMEM119, anti-GADPH, and anti-ZO-1 were purchased from Abcam (Cambridge, England). Anti-F4/80 was purchased from CST. GM-CSF was purchased from eBiosciences. Small EVs Spin Columns were obtained from Invitrogen Co. (California, USA). PEG-PLGA NPs was synthesized in our laboratory as previously described. Other chemicals were purchased from J&K (Beijing, China).
9
Neuro-Inflammatory Markers and Microbiome Analysis
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We purchased PB and permethrin from Sigma-Aldrich (St. Louis, MO). Anti-RAGE, anti-Claudin 5, anti-HMGB1, anti-IL-1β, and anti-ASC-2 were purchased from Santacruz Biotechnology (Dallas, TX), Anti-BDNF from cell signaling technology (Danvers, MA), while anti-NLRP3, anti-3-nitrotyrosine, anti-IL-6, anti-IL-18, anti-TMEM 119 primary antibodies were purchased from Abcam (Cambridge, MA). Species specific biotinylated conjugated secondary antibodies and Streptavidin-HRP (Vectastain Elite ABC kit) were purchased from Vector Laboratories (Burlingame, CA). Fluorescence conjugated (Alexa Fluor) secondary antibodies, ProLong Diamond antifade mounting media with DAPI and Pierce LAL chromogenic endotoxin quantitation kit were bought from Thermo Fisher Scientific (Waltham, MA) while enzyme-linked immunosorbent assay (ELISA) kits were purchased from ProteinTech (Rosemond, IL). Unless otherwise specified, all other chemicals used were purchased from Sigma. Paraffin-embedding of tissue and sectioning were done by AML laboratories (Baltimore, MD) and at the Instrument Resources Facility, University of South Carolina School of medicine (Columbia, SC). Microbiome analysis was done by Cosmos ID (Rockville, MD).
10
Spinal Cord Immune Cell Profiling
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Spinal cord sections were blocked with a blocking solution containing 5% non-fat milk powder, 1% bovine serum albumin and 0.3% Triton-X100 in 0.1 M PBS (all Sigma-Aldrich, USA) for 1 h at room temperature. The following primary antibodies, diluted in the same blocking solution, were then added and incubated at 4 °C overnight: Anti-Iba1 (1:200; Novus Biologicals, USA) for macrophages, anti-iNOS (1:100; Abcam, USA) for M1-macrophages, anti-CD206(1:200; Bio-Rad, Germany) for M2-macrophages, anti-TMEM119 (1:200; Abcam, USA) for microglia, anti-CD3 (1:200; Bio-Rad, Germany) for T-Lymphocytes and anti-GFAP (1:250; Abcam, USA) for astrocytes. Isotype controls with non-specific immunoglobulin at the same concentration were performed to ensure the specificity of the antibodies (data not shown).
As secondary antibodies, Alexa Fluor 405 donkey antigoat (1:400; Abcam, USA), Alexa Fluor 557 donkey antimouse (1:400; R&D Systems, USA) and Alexa Fluor 647 donkey anti-rabbit (1:400; Abcam, USA), diluted in blocking solution without Triton-X100, were used and applied for 1 h at room temperature before covering the sections with mounting medium.
As secondary antibodies, Alexa Fluor 405 donkey antigoat (1:400; Abcam, USA), Alexa Fluor 557 donkey antimouse (1:400; R&D Systems, USA) and Alexa Fluor 647 donkey anti-rabbit (1:400; Abcam, USA), diluted in blocking solution without Triton-X100, were used and applied for 1 h at room temperature before covering the sections with mounting medium.
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