Histological analysis of snap frozen tissue was performed as previously described47 (link). Antibodies against CD169 (Bio-Rad), CD11c, B220, CD90.2, F4/80, Ly6C (ebioscience), donkey-anti-rat secondary antibody (jackson immunoresearch), and self-made anti-LCMV monoclonal antibody (Clone: VL-4), were used. Images were acquired by ZEISS Axio Observer Z1.
Cd169
Manufactured by Bio-Rad
CD169 is a cell surface sialic acid-binding immunoglobulin-like lectin. It functions as a receptor involved in the binding and internalization of sialylated ligands.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Cd206, by Bio-Rad (2 mentions)
Axio observer z1, by Zeiss (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
F6397, by Bio-Rad (1 mentions)
Sytox blue nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Mca1218f, by Merck Group (1 mentions)
Sab4700700, by Bio-Rad (1 mentions)
Mca1971pe, by Bio-Rad (1 mentions)
Mca2311f, by Merck Group (1 mentions)
Mca2316f, by Merck Group (1 mentions)
F6397, by Southern Biotech (1 mentions)
Mca928pe, by Bio-Rad (1 mentions)
F4 80, by Santa Cruz Biotechnology (1 mentions)
Epifluorescence microscope, by Nikon (1 mentions)
Podoplanin, by R&D Systems (1 mentions)
Alexa568 donkey anti rat igg pab, by Abcam (1 mentions)
Dm5500b, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Cd45.1 a20, by Thermo Fisher Scientific (1 mentions)
6 protocols using cd169
1
Histological Characterization of Immune Cells
2
Multicolor Flow Cytometry Immunophenotyping
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Cells were blocked in PBS/2%FBS, on ice for 30 minutes. 2x10 5 cells/well were transferred to a 96-well V-bottomed plate and pelleted at 300 x g, 4 minutes, 4°C. The supernatant was removed by inverting the plate. The pellet was resuspended with 25 l of diluted, conjugated antibody and incubated in the dark, on ice for 30 minutes. The cells were then pelleted at 300 x g, 4 minutes, 4°C and washed twice with 75 l PBS. The final pellet was resuspended in 100 l PBS. 100l SYTOX Blue Nucleic Acid Stain (5M, ThermoFisher #S11348) was added immediately prior to flow cytometer analysis to allow for live/dead cell identification.
Antibodies used were CD14 (Biorad, #MCA1218F, 1:50) with isotype control (Sigma, #SAB4700700), CD16 (Biorad, #MCA1971PE, 1:200) with isotype control (Biorad, #MCA928PE), CD163 (Biorad, #MCA2311F, 1:100) with isotype control (Sigma, #F6397), CD169 (Biorad, #MCA2316F, 1:100) with isotype control (Sigma, #F6397) and CD172a (Southern Biotech, #4525-09, 1:400) with isotype control (Biorad, #MCA928PE).
Antibodies used were CD14 (Biorad, #MCA1218F, 1:50) with isotype control (Sigma, #SAB4700700), CD16 (Biorad, #MCA1971PE, 1:200) with isotype control (Biorad, #MCA928PE), CD163 (Biorad, #MCA2311F, 1:100) with isotype control (Sigma, #F6397), CD169 (Biorad, #MCA2316F, 1:100) with isotype control (Sigma, #F6397) and CD172a (Southern Biotech, #4525-09, 1:400) with isotype control (Biorad, #MCA928PE).
3
Multicolor Fluorescence Immunohistochemistry
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Organs were fixed in 1% formaldehyde / 10 mM sodium periodate / 75 mM L-lysine (18h, 4°C), equilibrated in 30% sucrose (24h, 4°C), then frozen in OCT. Sections (6μm) were air-dried (1h, 23°C), washed 3x in PBS, blocked with 0.3% Triton X-100 / 5% normal donkey serum (1h, 23°C), then incubated (18h, 4°C) with combinations of antibodies to eGFP (rabbit, chicken or goat pAb), CD68 (rat mAb, FA-11) (AbCam), B220 (rat mAb RA3-6B2), F4/80 (rat mAb CI:A3–1) (Santa Cruz Biotechnology), mCherry (rabbit pAb, Badrilla), CD206 (rat mAb MR5D3), CD169 (rat mAb 3D6.112) (Serotec), podoplanin (goat pAb, R&D Systems), and MuHV-4 (polyclonal rabbit sera raised by 2 subcutaneous virus inoculations). Sections were washed 3× in PBS, incubated (1h, 23°C) with combinations of Alexa568-donkey anti-rat IgG pAb, Alexa488 or Alexa647-donkey anti rabbit IgG pAb, Alexa647-donkey anti-mouse IgM pAb, Alexa488-donkey anti-chicken IgG pAb (Abcam), and Alexa488-donkey anti-goat pAb (Life Technologies), then washed 3× in PBS, stained with DAPI and mounted in Prolong Gold (Life Technologies). TdTomato fluoresence was visualized directly. Images were captured with a Zeiss LCM510 confocal microscope or a Nikon epifluorescence microscope and analyzed with Zen imaging software or ImageJ.
4
Immunofluorescence Microscopy of Frozen Tissues
5
Immunostaining of Midbrain Sections
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Midbrain sections (50 µm) were immunostained for anti-TH (EMD Millipore), which was followed by incubation with HRP-conjugated secondary antibody, and visualized by diaminobenzidine (DAB) staining. Sections were counterstained with thionin solution (Nissl stain).
Midbrain sections (50 µm) were blocked and permeabilized in PBS buffer containing 0.3% horse serum and 0.25% Triton X-100 for 1 h and then immunostained for 48 h with a combination of TH (EMD Millipore), CD11b (Abcam or eBioscience)/Iba1 (Wako Pure Chemical Industries), and P2Y12 (gift from D. Julius, University of California, San Francisco, San Francisco, CA)/CD169 (AbD Serotec) or combination of TH and CD11b (eBioscience). Adequate Alexa Fluor–conjugated secondary antibodies were used for detection by immunofluorescence microscopy. DNs were assessed as TH+. Resident microglia were identified as CD11b+P2Y12+ or Iba1+CD169− and infiltrating monocytes as CD11b+P2Y12− or Iba1+CD169+. In MPTP-injected tdTomatoR26f/f;Cx3cr1creER mice, infiltrating monocytes were assessed as CD11b+tdTomato− cells.
Midbrain sections (50 µm) were blocked and permeabilized in PBS buffer containing 0.3% horse serum and 0.25% Triton X-100 for 1 h and then immunostained for 48 h with a combination of TH (EMD Millipore), CD11b (Abcam or eBioscience)/Iba1 (Wako Pure Chemical Industries), and P2Y12 (gift from D. Julius, University of California, San Francisco, San Francisco, CA)/CD169 (AbD Serotec) or combination of TH and CD11b (eBioscience). Adequate Alexa Fluor–conjugated secondary antibodies were used for detection by immunofluorescence microscopy. DNs were assessed as TH+. Resident microglia were identified as CD11b+P2Y12+ or Iba1+CD169− and infiltrating monocytes as CD11b+P2Y12− or Iba1+CD169+. In MPTP-injected tdTomatoR26f/f;Cx3cr1creER mice, infiltrating monocytes were assessed as CD11b+tdTomato− cells.
6
Multicolor Immunofluorescence Staining of Organs
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The organs were fixed in 1% formaldehyde–10 mM sodium periodate–75 mM l -lysine (18 h, 4°C), equilibrated in 30% sucrose (18 h, 4°C), and then frozen. Sections were blocked with 0.1% Triton X-100–5% normal goat serum and then incubated (18 h, 4°C) with antibodies to B220 (rat MAb RA3-6B2), surfactant protein C (SPC) (goat polyclonal antibody [pAb]), CK19 (goat MAb N-13; Santa Cruz Biotechnology), CD68 (rat MAb FA-11), ER-TR7 (rat MAb), CD31 (rat MAb MEC 7.46), β-galactosidase (chicken pAb), LYVE-1 (rabbit pAb; Abcam), podoplanin (PDP) (goat pAb, R&D Systems), CD11c (hamster MAb HL-3; BD Pharmingen), CD206 (rat MAb MR5D3) and CD169 (rat MAb 3D6.112) (Serotec), peripheral node addressin (PNAd) (rat MAb MECA-79; BioLegend), or aquaporin V (rabbit pAb; Alamone Labs). After incubation with primary antibodies, the sections were washed three times in phosphate-buffered saline (PBS), incubated (1 h, 23°C) with combinations of Alexa Fluor 488-, Alexa Fluor 568-, or Alexa Fluor 647-conjugated goat pAb (Abcam), then washed three times in PBS, stained with 4′,6′-diamidino-2-phenylindole (DAPI), and mounted in ProLong gold (Life Technologies). TdTomato (Tom) and GFP expression were visualized directly. Images were acquired with a Zeiss LSM510 microscope and analyzed with Zen software.
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