After 20 days of treatment using 30 mg/kg/day of masitinib, starting after paralysis onset, animals were deeply anesthetized and transcardial perfusion was performed with 0.9 % saline and 4 % paraformaldehyde in 0.1 M PBS (pH 7.2–7.4). Fixed spinal cord was removed, post-fixed by immersion for 24 h, and then transverse sectioned (30 μm) in a Leica cryostat. Serial sections were collected in 100 mM PBS for immunohistochemistry. Free-floating sections were permeabilized for 30 min at room temperature with 0.3 % Triton X-100 in PBS, passed through washing buffered solutions, blocked with 5 % BSA:PBS for 1 h at room temperature, and incubated overnight at 4 °C in a solution of 0.3 % Triton X-100 and PBS containing the primary antibodies, rabbit anti-GFAP (1:500, Sigma), mouse anti-S100β (1:400, Sigma), rabbit anti-Iba1 (1:300, abcam), rabbit anti-CD206 (1:300 abcam), mouse anti-CD68 (1:200, abcam), mouse anti-ChAT (choline acetyltransferase) (1:300, Millipore). After washing, sections were incubated in 1:1000-diluted secondary antibodies conjugated to Alexa Fluor 488 and/or Alexa Fluor 633 (1:1000, Invitrogen). Antibodies were detected by confocal microscopy using a confocal LEICA TCS-SP5-DMI6000 or a confocal ZEISS LSM 780.
Rabbit anti cd206
Manufactured by Abcam
Sourced in United States, United Kingdom
Rabbit anti-CD206 is a primary antibody that recognizes the CD206 antigen, also known as the macrophage mannose receptor. CD206 is a C-type lectin receptor expressed on the surface of macrophages and dendritic cells, and plays a role in the clearance of glycoproteins. This antibody can be used for the detection and characterization of CD206-expressing cells in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti inos, by Abcam (7 mentions)
Mouse anti cd68, by Abcam (4 mentions)
Rabbit anti cd86, by Abcam (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Mouse anti f4 80, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti iba1, by Fujifilm (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Rat anti cd68, by Bio-Rad (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Rabbit anti iba1, by Abcam (1 mentions)
Mouse anti chat, by Merck Group (1 mentions)
Rabbit anti gfap, by Merck Group (1 mentions)
Alexa fluor 633, by Thermo Fisher Scientific (1 mentions)
Mouse anti s100β, by Merck Group (1 mentions)
Cryostat, by Leica camera (1 mentions)
22 protocols using rabbit anti cd206
1
Masitinib Treatment in Spinal Cord Injury
2
Immunofluorescence Staining of Immune Cells
3
Immunohistochemical Analysis of CD3 and CD206 in Ischemic Brain
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Six serial coronal sections (10 μm thick) per animal were collected at six regions: bregma + 0.06, − 0.78, − 1.2, − 1.62, − 2.04, − 2.46 mm. Frozen brain sections (10 μm) were fixed in 4% paraformaldehyde for 15 min and washed in 0.01 M PBS (3 × 10 min). Sections were then blocked with 10% goat serum (Sigma) for 60 min to block non-specific binding of the secondary antibody. Sections were then incubated overnight at 4 °C with either rabbit anti-CD3 (1:200; Abcam) or rabbit anti-CD206 (1:500; Abcam). On the following day, they were washed (PBS; 3 × 10 min) and incubated for a maximum of 2 h with either goat anti-rabbit Alexa Fluor 594 (1:500; Thermofisher Scientific) or goat anti-rabbit Alexa Fluor 488 (1:500; Thermofisher Scientific). Finally, sections were again washed and then mounted with Vectashield medium containing 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories), and a coverslip was applied. All tissue-mounted slides were viewed, analyzed and photographed with an Olympus fluorescence microscope. Numbers of immunoreactive cells were counted manually per whole ischemic hemisphere and then averaged across the six regions, as indicated above.
4
Immunofluorescence Staining of Brain Sections
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After washed with PBS (5 min each). Prepared brain sections were permeated with 0.5% Triton X-100 for 30 min, and blocked with 10% goat serum for 2 h at room temperature, followed by overnight incubation with primary antibody at 4°C. The next day, samples were incubated with the corresponding secondary antibody at room temperature for 2 h. And 4′, 6-diamino-2-phenylindole (DAPI; Boster, Wuhan, China) were counterstained for 5 min. Finally, stained sections were examined, and images were captured using a confocal microscope (LSM-880; Zeiss). The primary antibodies used in the experiment are as follows: goat anti-ionized calcium-binding adaptor molecule (Iba1; 1:200; Abcam, UK), rabbit anti-Iba1 (1:200; GeneTex, Irvine, CA, USA), mouse anti-CD86 (1:200; Santa Cruz, CA, USA), rabbit anti-CD206 (1:200; Abcam, UK). Images from four sections of the brain around the PAG, VTA and VPL were captured using a 20× objective on a Zeiss confocal microscope (Zeiss, LSM780, Germany). Cell numbers were calculated per random microscopic field (100 × magnification). All counts were performed in a blinded fashion.
5
Bladder Macrophage Characterization in Rats
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Bladders were isolated from the rats in all groups and fixed in 4% paraformaldehyde. The tissues were frozen and cut on a cryostat at 5-μm thickness. Sections were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature. The slides were incubated with mouse anti-F4/80 (1:50; Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit anti-iNOS (1:100; Abcam plc, Cambridge, UK), or rabbit anti-CD206 (1:500; Abcam plc) overnight at 4°C and then with anti-mouse Alexa Fluor Plus 594 (1:1000; Abcam plc), anti-rabbit Alexa Fluor Plus 488 (1:1000; Thermo Fisher Scientific, Waltham, MA, USA) secondary antibodies, and 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) for 1 h at room temperature. Immunofluorescence images were obtained using an all-in-one fluorescence microscope (BZ-X800; KEYENCE) and analyzed using a BZ-X800 analyzer. Two random areas per bladder selection from each group were observed and the macrophage in the lamina propria was estimated within the visual fields observed at object lens 10× magnification. The percentage of iNOS- or CD206-positively stained cells per F4/80-positively stained cells in the fields was determined.
6
Antibody Characterization for Cellular Analysis
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The following antibodies were used: mouse anti-F4/80 (Santa Cruz), rabbit anti-iNOS (Cell Signaling Technology), mouse anti-CD80 (Invitrogen Antibodies), rabbit anti-CD206 (Abcam), rabbit anti-SHP2 (Cell Signaling Technology), rabbit anti-COL2 (Abcam), rabbit anti-COL10 (Abcam), rabbit anti-MMP3 (Proteintech), rabbit anti-COX2 (Cell Signaling Technology), rabbit anti-GAPDH (Cell Signaling Technology), rabbit anti-p-P65 (Cell Signaling Technology), rabbit anti-P65 (Cell Signaling Technology), rabbit anti-β-actin (Cell Signaling Technology), rabbit anti-histone H3 (Cell Signaling Technology), rabbit anti-p-AKT (Cell Signaling Technology), rabbit anti-AKT (Cell Signaling Technology), and rabbit anti-p-IKKα/β (Cell Signaling Technology).
7
Immunofluorescence Analysis of Brain Tissue
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The paraffin section (5 μm-thick) of the brain was prepared, after being fixed, washed, permeabilized, and blocked, cells were incubated with rabbit anti-ZO-1 (1:200, Abcam), or rabbit anti-Iba-1 (1:100, Proteintech). After that, sections were further incubated with the fluorescein 488-conjugated anti-rabbit antibody (1:1000, Jackson Immunoresearch). Slides were imaged using a fluorescence microscope. To explore the polarization reprogramming effects of DEX, we performed immunofluorescence double labeling. Sections were incubated in mouse anti-Iba-1 (1:100, Abcam), with rabbit anti-iNOS (1:100, Abcam) or rabbit anti-CD206 (1:100, Abcam) for 1 h at 37°C. Antibodies were then used: the fluorescein 488-conjugated anti-rabbit antibody (1:1000, Jackson Immunoresearch, United States), or Cy3-conjugated anti-mouse antibody (1:1000, Jackson Immunoresearch).
8
Immunofluorescence Staining of Vascular Cells
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Immunofluorescence staining was performed as previously described [5 (link)]. Smooth muscle cells were stained using mouse anti-SM-MHC (Abcam, UK) primary antibodies. Endothelial cell staining was performed using rabbit anti-vWF (Abcam, USA) primary antibody. The vascular precursor cell was stained using rabbit anti-Sca-1 (Millipore, Germany). For elastin and collagen staining, slides were incubated with rabbit anti-Elastin (Abcam, UK), mouse anti-Collagen I (Abcam, UK) and mouse anti-Collagen III (Abcam, UK). To observe inflammatory cells in the explanted grafts, mouse anti-CD68 (Abcam, UK), rabbit anti-iNOS (Abcam, UK) and rabbit anti-CD206 (Abcam, UK) were used as primary antibodies. After overnight incubated at 4 °C, slides were washed twice with PBS solution and incubated with the respective fluorescein isothiocyanate-conjugated secondary antibodies for 60 min at 37 °C. The samples were observed using an Olympus Flu view 1000 confocal microscope (Japan). Tissue slides pretreated without a primary antibody were used as negative controls. Cell populations were determined based on cell counts from each image on six different parts (12, 2, 4, 6, 8, and 10 o'clock positions). Data were collected from three different samples in each group. Details of the primary antibodies are listed in Table S1 .
9
Histological and Immunohistochemical Analysis of Osteoarthritis in Mice
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The right knee joints of mice were fixed in 4% buffered paraformaldehyde for two days. Then, the samples were decalcified in 10% ethylenediaminetetraacetic acid solution for three weeks. The paraffin blocks were coronally cut into sections with 6 μm thickness. The sections were deparaffinized, dehydrated, and processed for hematoxylin and eosin (H&E) and Safranin O and Fast Green staining. Immunohistochemistry was performed according to a standard protocol. The sections were separately incubated with mouse anti-COL2A1 (Santa Cruz Biotechnology, Texas, USA, 1 : 50), rabbit anti-MMP-13 (Bioss, Beijing, China, 1 : 200), rabbit anti-F4/80 (Cell Signaling, 1: 400), rabbit anti-iNOS (Abcam, 1 : 100), and rabbit anti-CD206 (Abcam, 1 : 1000) overnight at 4°C. Then, the slides were incubated with a biotin-labeled IgG secondary antibody (Zhongshan Golden Bridge, Beijing, China) and stained with a diaminobenzidine (DAB) substrate and counterstained with hematoxylin. Cartilage degradation and synovitis were evaluated using the Osteoarthritis Research Society International (OARSI) scoring system by two independent observers blinded to treatment [19 (link)].
10
Candesartan Modulates Microglial Phenotype
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BV2 microglial cells (1.5 × 104 cells/well) were seeded in a 24-well
plate containing the coverslip. The cells were treated with LPS (100 ng/mL) +
IFN-γ (20 ng/mL) followed by vehicle or selected dose of candesartan. After 24
h, cells were fixed with 4% paraformaldehyde followed by blocking with 5% donkey
serum. The cells were incubated with primary antibodies, rabbit anti-iNOS (1:100
dilution, Abcam) or rabbit anti-CD206 (1:200 dilution, Abcam) at 4°C overnight.
Cells were washed with PBS, followed by incubation with the secondary antibody
for 2 h at room temperature, Alexa Fluor 488 donkey anti-rabbit IgG (1:400
dilution, Invitrogen). After being washed, the nucleus was stained with DAPI.
All samples were observed with a laser confocal microscope (Nikon).
plate containing the coverslip. The cells were treated with LPS (100 ng/mL) +
IFN-γ (20 ng/mL) followed by vehicle or selected dose of candesartan. After 24
h, cells were fixed with 4% paraformaldehyde followed by blocking with 5% donkey
serum. The cells were incubated with primary antibodies, rabbit anti-iNOS (1:100
dilution, Abcam) or rabbit anti-CD206 (1:200 dilution, Abcam) at 4°C overnight.
Cells were washed with PBS, followed by incubation with the secondary antibody
for 2 h at room temperature, Alexa Fluor 488 donkey anti-rabbit IgG (1:400
dilution, Invitrogen). After being washed, the nucleus was stained with DAPI.
All samples were observed with a laser confocal microscope (Nikon).
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