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Goat anti cd206

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Goat anti-CD206 is a laboratory reagent used for the detection and analysis of the CD206 protein, also known as the macrophage mannose receptor. This antibody is produced in goats and can be used in various immunological techniques, such as flow cytometry and immunohistochemistry, to identify and study cells expressing the CD206 receptor.

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27 protocols using goat anti cd206

1

Comprehensive Immunostaining Protocol

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Immunofluorescence staining analysis was performed on 40 μm free-floating sections. Primary antibodies, including chicken anti-GFP (Aves Labs, GFP-1020), goat anti-CD206 (R&D Systems, AF2535), rabbit anti-Cleaved Caspase-3 (Cell Signaling, 9661), mouse anti-Caspase-3 (Novus Biologicals, 31A1067), mouse anti-NeuN (Millipore, MAB377), rabbit anti-Tmem119 (Abcam, ab209064), mouse anti-LPL (Abcam, ab21356), rabbit anti-CX3CR1(Abcam, ab8021), rabbit anti-GFAP (Abcam, ab7260), mouse anti-iNOS (inducible nitric oxide synthase) (Abcam, ab49999), and goat anti-Iba1 (Abcam, ab5076), were used. Corresponding secondary antibodies, including 488- and 594-conjugated secondary antibodies, were purchased from Thermo Fisher Scientific, Alexa Fluor conjugates.
All images were processed with Image J for quantification analysis. The means were calculated from 3 randomly selected microscopic fields in the ipsilateral and contralateral cortex of each section, respectively, and 3 consecutive sections were analyzed for each brain. Data are expressed as mean numbers of cells per square millimeter.
Ye S.Y., Apple J.E., Ren X., Tang F.L., Yao L.L., Wang Y.G., Mei L., Zhou Y.G, & Xiong W.C. (2019). Microglial VPS35 deficiency regulates microglial polarization and decreases ischemic stroke-induced damage in the cortex. Journal of Neuroinflammation, 16, 235.
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2

Immunohistochemical Analysis of Microglia

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The brain sections and fixed cells were placed in 3% H2O2/methyl alcohol for 30 min and then placed in blocking solution for 30 min. The brain sections and cells were incubated with primary antibodies (rat anti-CD16/CD32 (1:500); BD Pharmingen, BD Biosciences; goat anti-CD206 (1:200), R&D Systems; rabbit anti-ionised calcium-binding adaptor molecule 1 (Iba-1) (1:200), Wako Chemicals, Virginia, USA) overnight at 4°C and immersed in a secondary antibody solution (Alexa Fluor 488-conjugated anti-mouse, Alexa Fluor 594-conjugated anti-goat and Alexa Fluor 594-conjugated anti-rat antibodies (1:200), Life Sciences, Paisley, UK) for 2 hours. For TUNEL (Terminal dexynucleotidyl transferase(TdT)-mediated dUTP Nick End Labeling) staining, the brain slices were incubated in working liquid (Roche) for 1 hour after blocking with 3% donkey serum. The brain sections and cells were covered with antifade medium containing DAPI (4',6-diamidino-2-phenylindole) images were acquired with a laser scanning confocal microscope (Nikon Ti-E, Japan). The number of positively stained cells per square millimetre was determined with NIH ImageJ software (Bethesda, Maryland, USA) by an investigator blinded to the experimental groups.
Yan F., Cheng X., Zhao M., Gong S., Han Y., Ding L., Wu D., Luo Y., Zuo W., Zhu L., Fan M, & Ji X. (2021). Loss of Wip1 aggravates brain injury after ischaemia/reperfusion by overactivating microglia. Stroke and Vascular Neurology, 6(3), 344-351.
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3

Quantitative Immunohistochemical Analysis of White Matter Injury

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Immunostaining was performed on free-floating cryosections. Sections were incubated in the following primary antibodies overnight at 4 °C: rabbit anti-myelin basic protein (MBP; Abcam), mouse anti-nonphosphorylated neurofilament H (SMI32; Calbiochem), rabbit anti-Iba1 (Wako), goat anti-CD206 (R&D Systems), rat anti-CD16 (BD biosciences), mouse anti-BrdU (BD bioscience), rabbit anti NG2 (Millipore), and mouse anti-adenomatous polyposis coli protein (APC; Calbiochem). For BrdU staining, brain sections were pretreated with 1N HCl for 1 hour followed by 0.1 mol/L boric acid (pH 8.5) for 10 min at 37 °C. Images were captured using a confocal laser scanning microscope (Olympus Fluoview FV1000; Olympus). The immunostaining intensity with MBP and SMI32 antibodies, as well as the numbers of target immunopositive cells were quantified by a blinded investigator using Image J software. Three randomly selected microscopic fields within the external capsule, cortex, striatum, subventricular zone (SVZ), and subgranular zone (SGZ) on each of 3 consecutive sections were analyzed for each brain by a blinded investigator. White matter injury was expressed as the mean ratio of SMI32 to MBP immunostaining. Immunopositive cell counts were presented as the mean number of cells per square millimeter.
Han L., Cai W., Mao L., Liu J., Li P., Leak R.K., Xu Y., Hu X, & Chen J. (2015). Rosiglitazone promotes white matter integrity and long-term functional recovery after focal cerebral ischemia. Stroke; a journal of cerebral circulation, 46(9), 2628-2636.
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4

Immunofluorescence Staining of Lymphatic and Vascular Markers

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Tail skin and diaphragms were fixed in fresh 4 % paraformaldehyde (PFA) for 2 h and blocked at room temperature for 3 h in phosphate-buffered saline (PBS) containing 5 % donkey serum, 1 % bovine serum albumin (BSA) and 0.1 % Triton-X. The following antibodies were incubated overnight: Rabbit anti-LYVE-1 (AngioBio), goat anti-LYVE-1 (R&D Systems), rat anti-CD31 (BD Biosciences Pharmingen), goat anti-CD206 (R&D Systems), mouse anti-smooth muscle actin (Sigma-Aldrich), rat anti-CD68 (Abcam), goat anti-Prox-1 (R&D Systems), rabbit anti-Prox-1 (kind gift of Dr. Kari Alitalo), rat anti-F4/80 (Abcam) and rat anti-CSF-1R (AFS98 hybridoma antibody). After an intense washing step, tissues were incubated for 2 h at room temperature with Alexa Fluor 488, 594 or 647 nm-conjugated secondary antibodies (Invitrogen) and mounted in Vectashield (Vector) for confocal imaging. Whole-mount z-stack images were acquired with a LSM 710 FCS confocal microscope and ZEN software (Zeiss) and processed with Photoshop CS5 (Adobe) and ImageJ software (NIH).
Ochsenbein A.M., Karaman S., Proulx S.T., Goldmann R., Chittazhathu J., Dasargyri A., Chong C., Leroux J.C., Stanley E.R, & Detmar M. (2016). Regulation of lymphangiogenesis in the diaphragm by macrophages and VEGFR-3 signaling. Angiogenesis, 19(4), 513-524.
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5

Immunofluorescence Staining of BV2 Microglia

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BV2 microglia were fixed in 4% paraformaldehyde for 15 min. After rinses in PBS, the cells were incubated with the following primary antibody: rat anti-CD16/32 (BD Biosciences Pharmingen, CA, USA) and goat anti-CD206 (R&D Systems, MN, USA) overnight at 4 °C, followed by secondary antibodies for 1 h with extensive washing. Images were captured by Nikon Optical TE2000-S inverted fluorescence microscope.
Du R.H., Sun H.B., Hu Z.L., Lu M., Ding J.H, & Hu G. (2018). Kir6.1/K-ATP channel modulates microglia phenotypes: implication in Parkinson’s disease. Cell Death & Disease, 9(3), 404.
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6

Immunofluorescence Staining of Brain Slices

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Brain slices were obtained and used for immunofluorescence staining, as previously described (Ma et al. 2016 (link)). The primary antibodies were goat anti-CD206 (1:200; R&D Systems, Minneapolis, MN, USA), rat anti-CD16/32 (1:100; BD Pharmingen, San Jose, CA, USA) and rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) (1:200; Wako Pure Chemical Industries, Osaka, Japan). The fluorophore-conjugated secondary antibodies (Invitrogen Corporation, Carlsbad, CA, USA) used were donkey anti-goat antibody conjugated to Alexa 488 (1:200), donkey anti-rat antibody conjugated to Alexa 594 (1:200) and goat anti-rabbit antibody conjugated to Alexa 594 (1:200). Images were captured using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Mean merged cell counts were calculated from three randomly selected microscope fields in the cortex of each section, and three consecutive sections were analyzed from each brain. All images were manually counted. Data are expressed as mean number of cells/mm2. The person evaluating TUNEL staining and immunofluorescence staining was blinded to the treatment group of the mice.
Li F., Ma Q., Zhao H., Wang R., Tao Z., Fan Z., Zhang S., Li G, & Luo Y. (2018). L-3-n-Butylphthalide reduces ischemic stroke injury and increases M2 microglial polarization. Metabolic Brain Disease, 33(6), 1995-2003.
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7

Immunofluorescence Markers for Microglia and Macrophages

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Antibodies used were rabbit anti-Iba1 (WAKO, AB_839504, 1:600), rat anti-mouse CD68 (Bio-rad, AB_322219, 1:500), rat anti-CD86 (BD Pharmingen, AB_394994, 1:100), goat anti-CD206 (anti mMMR, R&D Systems, AB_2063012, 1:00), goat anti-YM1 (R&D Systems, AB_2079008, 1:250), and rat anti-mouse MARCO (Bio-rad, AB_322923, 1:500). Secondary Antibodies from Jackson Labs: Donkey anti-Rabbit Alexa Fluor 647, AB_2492288; Donkey anti-mouse Alexa Fluor 488, AB_2340846; Donkey anti-rat Alexa Fluor 488, AB_2340683; Donkey anti-goat Alexa Fluor 488, AB_2340428.
Brown E.V., Falnikar A., Heinsinger N., Cheng L., Andrews C.E., DeMarco M, & Lepore A.C. (2021). Cervical spinal cord injury-induced neuropathic pain in male mice is associated with a persistent pro-inflammatory macrophage/microglial response in the superficial dorsal horn. Experimental neurology, 343, 113757.
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8

Multimarker Immunohistochemistry of the Brain

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Animals were deeply anesthetized before intracardiac perfusion with 0.9% saline followed by 4% paraformaldehyde. Brains were cryoprotected in 30% sucrose in PBS. Immunohistochemistry was performed on serial coronal sections (4–5 sections per animal per stain). Primary antibodies include the following: goat anti-CD206 (1:200, R&D Systems), rat anti-CD16/32 (1:500, BD), rabbit anti-myelin basic protein (MBP, 1:200, Abcam), mouse anti-non-phosphorylated neurofilament H (SMI-32, 1:1000, Calbiochem) and rabbit anti-ionized calcium binding adaptor molecule 1 (Iba1, 1:1000, Wako). Sections were also stained for myelin using Luxol fast blue.
Chen S., Zhang H., Pu H., Wang G., Li W., Leak R.K., Chen J., Liou A.K, & Hu X. (2014). n-3 PUFA supplementation benefits microglial responses to myelin pathology. Scientific Reports, 4, 7458.
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9

Isolation and Characterization of Single Brain Cells

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Single brain cells were prepared as mentioned. Four ischemic hemispheres were pooled and stained using Biotin-anti-Ly6G (Biolegend) with 1ug antibodies per 106 cells. Cells were washed with PBS and further stained with anti-Biotin microbeads (Miltenyi Biotec) for 1ug antibodies per 106 cells. After two washes with PBS, cells were subjected to positive selection program using autoMACS (Miltenyi Biotec). Cells were washed and then stained with goat anti-CD206 (1:500, R&D system) or rabbit anti-MPO (1:500, R&D system) for 30min at room temperature. After 3 washes, cells were dropped on slides and mounted with Fluoromount-G (Southern Biotech).
Cai W., Liu S., Hu M., Huang F., Zhu Q., Qiu W., Hu X., Colello J., Zheng S.G, & Lu Z. (2019). Functional dynamics of neutrophils after ischemic stroke. Translational stroke research, 11(1), 108-121.
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10

Immunofluorescence Staining of Brain Slices

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Brain slices were prepared as previously described,
21 (link) and 0.3% Triton ×100 was used to penetrate 15 min. Brain slices (30 μm) were blocked with QuickBlock™ (Beyotime, China) for 1 h, and then incubated in a refrigerator with primary antibodies at 4°C overnight. We performed single immunofluorescence staining using rabbit anti‐Iba1 (1:1000, Abcam, USA). Performed double immunofluorescence staining using mouse anti‐Iba1 (1:100, Santa Cruz Biotechnology, USA) with rabbit anti‐TRPV4 (1:200, Abcam, USA), rabbit anti‐Iba1 (1:1000, Abcam, USA) with mouse anti‐CD86 (1:50, Santa Cruz Biotechnology, USA), and rabbit anti‐Iba1 (1:1000, Abcam, USA) with goat anti‐CD206 (1:250, R&D Systems, USA). After washing the brain slices with PBS containing 0.3% Triton ×100, Alexa 594‐conjugated goat anti‐rabbit antibodies (1:1000, Life Technologies Corporation, USA), Alexa 555‐conjugated donkey anti‐goat antibodies (1:1000, Life Technologies Corporation, USA), and Alexa 488‐conjugated goat anti‐rabbit antibodies (1:1000, Life Technologies Corporation, USA) were added and incubated at room temperature for 2 h. The nucleus was stained with DAPI. Using laser scanning confocal microscopy (Leica SP‐8) to take pictures. ImageJ was used for analysis.
Ren X., Gao X., Li Z., Ding Y., Xu A., Du L., Yang Y., Wang D., Wang Z, & Shu S. (2024). Electroacupuncture ameliorates neuroinflammation by inhibiting TRPV4 channel in ischemic stroke. CNS Neuroscience & Therapeutics, 30(2), e14618.
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