Sections were analyzed for three markers of the M1 phenotype (TNF-alpha, iNOS, and CCR7) and three markers of the M2 phenotype (CD206, Arg1, and CD163), along with the pan-macrophage marker F480, using the antibodies and dilutions described in [16 (link)] and CD163 (1:50) from Santa Cruz Biotechnology. Endothelial cells were stained with rabbit-anti-mouse CD31 (1:50) from Abcam.
Cd163
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
CD163 is a cell surface receptor expressed on the surface of monocytes and macrophages. It functions as a scavenger receptor, involved in the clearance of hemoglobin-haptoglobin complexes.
Automatically generated - may contain errors
Lab products found in correlation
F4 80, by Abcam (3 mentions)
Cd11b, by Abcam (2 mentions)
Gap43, by Novus Biologicals (2 mentions)
Cellquest software, by BD (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Alexa fluor 488, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Cy3 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ihc kit, by ZSGB-BIO (1 mentions)
P egfr, by Santa Cruz Biotechnology (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Rabbit anti mouse cd31, by Abcam (1 mentions)
Igfbp 2, by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Gap43, by Abcam (1 mentions)
Mertk, by R&D Systems (1 mentions)
Cytomics fc500 mlp, by Beckman Coulter (1 mentions)
Cd274, by Thermo Fisher Scientific (1 mentions)
20 protocols using cd163
1
Macrophage Phenotype Characterization
2
Muscle Fiber Characterization in Mice and Rats
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Primary antibodies used on mouse muscle included eMHC (Developmental Studies Hybridoma Bank [DSHB], F1.652s, 1:20), Pax7 (DSHB, Pax7c, 1:100), MHC I (DSHB, BA-D5c, 1:100), MHC IIa (DSHB, SC-71c, 1:100), MHC IIb (DSHB, BF-F3c, 1:100), laminin (Abcam, ab7463, 1:200), Ly6G (Gr-1) (BD Biosciences, BD550291, 1:50), CD68 (Bio-Rad, MCA1957, 1:50), and CD163 (Santa Cruz Biotechnology, sc-33560, 1:50). Primary antibodies used on rat muscle included MHC type I (DSHB, BA-D5c, 1:100), MHC IIa (DSHB, SC-71c, 1:100), MHC IIb (DSHB, BF-F3c, 1:100), HIS48 (Abcam, Ab33760, 1:20), CD68 (Abcam, ab31630, 1:50), and CD163 (Santa Cruz Biotechnology, sc-33560, 1:50). Antibody binding was visualized with standard Alexa Fluor secondary antibodies (Invitrogen, Thermo Fisher Scientific, 1:500 in PBS), except for Pax7, which was detected using a Tyramide SuperBoost Kit (Invitrogen, Thermo Fisher Scientific B40913). Fluorescent dyes DAPI (Invitrogen, Thermo Fisher Scientific, D21490, 2 μg/mL), wheat germ agglutinin (WGA) Alexa Fluor 647 conjugate (Invitrogen, Thermo Fisher Scientific, W32466, 5 μg/mL), WGA CF405S conjugate (Biotium, 29027, 100 μg/mL), and phalloidin (Invitrogen, Thermo Fisher Scientific ActinRed 555 ReadyProbes, R37112) were used to counterstain cell nuclei, extracellular matrix, and muscle fibers, respectively.
3
Immunophenotyping of NR8383 Macrophages
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NR8383 cells were incubated with rat monoclonal primary antibody (CD11b, 1:100; Abcam, Cambridge, UK) followed by the appropriate secondary antibody (Cy3-conjugated secondary antibody, 1:350; Jackson ImmunoResearch Europe Ltd., Newmarket, UK). Cell surface glycoproteins CD163 (1:50; Santa Cruz Biotechnology, Dallas, TX, USA), CD44 (1:150; Novus Biologicals, Centennial, CO, USA) on fixed NR8383 cells were stained with rat monoclonal antibodies, followed by Alexa Fluor® 488 (1:350, Jackson ImmunoResearch). Injected human MSCs were detected with an anti-human β2 microglobulin (hβ2MG) antibody (1:100, Santa Cruz, Dallas, TX, USA) with an Alexa Fluor® 488-conjugated secondary antibody (1:350, Jackson ImmunoResearch). Before co-culturing with human MSCs, NR8383 cell nuclei were counterstained with Hoechst 33342 to prevent the staining of human nuclei. Fluorescent images were acquired and analyzed using an LSM 800 confocal microscope (Zeiss, Oberkochen, Germany).
4
Multimarker Immunohistochemistry Analysis
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Tissues were fixed in 4% paraformaldehyde, sent for sectioning, and assessed with an IHC kit (ZSGB-BIO, Beijing, China). Slides were incubated at 4°C overnight (16−20 h) with antibodies reactive with Ki67 (#ab16667; Abcam), F4/80 (#ab100790; Abcam), CD163 (#sc58965; Santa Cruz), VEGF (#ab32152; Abcam), VEGFR (#ab32152; Abcam), CD34 (#ab81289; Abcam), EGFR (#sc373746; Santa Cruz), and p-EGFR (#sc-377547; Santa Cruz). Diaminobenzidine (K176810E; ZSGB-BIO, China) was used for target protein staining and H&E for nuclear staining. Images were photographed at 400× with a microscope.
5
IHC and Flow Cytometry Analysis of PDAC Tumor Samples
6
Immunohistochemical Analysis of Orthotopic PDXs
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Orthotopic PDXs were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS; Gibco) and embedded in paraffin. Sectioned slides were blocked with 10% horse serum and permeabilized with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). Samples were probed with primary antibodies against the following proteins: CD68 and YKL-40 (Abcam, Cambridge, UK), CD163 (Santa Cruz Biotechnology, Dallas, TX, USA), oligodendrocyte TF 2 (R&D Systems, Minneapolis, MN, USA), CD44 (Sigma-Aldrich), B cell lymphoma 2-related protein A1 and Top2A (LSBio, Seattle, WA, USA), and serpin family E member 1 (Novus Biologicals, Centennial, CO, USA). Immunoreactivity was quantified using a Tissue FAXS system (Tissuegnostics USA, Tarzana, CA, USA). Scanned images were analyzed with HistoQuest cytometry software. The threshold signal intensity was determined relative to the negative control.
7
Spinal Cord Immune Cell Analysis
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The spinal cord tissue sections were treated with xylene and graded alcohol, and heated in citric acid for antigen retrieval. After blocking with 10% fetal bovine serum (FBS; Gibco, Life Technologies, United States) for 1 h at RT, the sections were incubated overnight with primary antibodies against ionized calcium binding adaptor molecule 1 (Iba-1, 1:200, and CST), CD68 (1:300, Boster Biological Engineering Co.), CD163 (1:30, Santa Cruz), GFAP (1:600, Abcam), GAP43 (1:200, Abcam) and MBP (1:200, Abcam) at 4°C. The following day, the sections were incubated with fluorochrome-conjugate rabbit or mouse secondary antibodies (1:300, Invitrogen), and mounted with DAPI medium (Solarbio). The sections were observed under a fluorescence microscope (Olympus IX73). The fluorescence intensity of the positively stained area was measured using ImageJ, and the average fluorescence intensity in each field of view was calculated.
8
Immunostaining of Primary Neuronal Cultures
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The primary cortical neurons cultured on the coverslips were fixed at defined time points with 4% PFA for 2 h at 4°C. The sections were dewaxed in xylene three times each for 5 min and re-hydrated through a series of alcohol with descending concentrations (100 to 95% to 80 to 70% and then tap water twice, each step for 5 min). The prepared tissue sections and cells were blocked in PBS with 10% normal horse serum at room temperature for 1 h and then incubated at 4°C overnight with the following primary antibodies in 10% normal horse serum: microtubule-associated protein 2 (MAP2, 1:200, Boster Biological Engineering Co.), Tuj1 (1:200, Millipore), GFAP (1:500, Boster Biological Engineering Co.), GAP43 (1:200, NOVUS), MBP (1:200, NOVUS), ionized calcium binding adaptor molecule 1 (Iba-1, 1:200, CST), CD163 (1:30, Santa Cruz), and CD68 (1:300, Boster Biological Engineering Co.). The secondary antibodies conjugated with Alexa fluor® fluorochrome (1: 300) were used to detected corresponding primary antibodies. The immunostaining results were checked under a fluorescence microscope (Olympus IX73).
9
Flow Cytometry Analysis of Cell Surface Proteins
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To determine the expression of the cell surface proteins, 1 × 106 cells were seeded in each culture dish (the samples were repeated three times in each group). After the differentiation period, the cells were incubated with primary antibodies for 30 min at 4 °C, washed thrice with DPBS, and then incubated for 30 min with PE-conjugated or FITC-conjugated secondary antibodies at 4 °C. After washing, the cells were re-suspended in 0.4 mL DPBS and analyzed using a Cytomics FC500 MLP (Beckman Coulter Inc., Fullerton, CA, USA). For measurement of the intracellular protein expression, the cells were fixed by incubation with 4% formaldehyde in DPBS for 10 min, followed by permeabilization with 0.1% Triton X-100 for 10 min at room temperature prior to staining with the primary antibodies, and then with PE-conjugated secondary antibodies. The following antibodies were used for flow cytometry: CD163 and CD206 from Santa Cruz Biotechnology (Dallas, TX, USA); CD209 from Serotec (Bio-Rad, Hercules, CA, USA); CD274 from eBioscience Inc. (San Diego, CA, USA); MER-TK from R&D Systems (Minneapolis, MN, USA); DHRS9 from Abnova (Taiwan, China); and TREM2 from Biolegend (San Diego, CA, USA).
10
Multimarker Immunofluorescence Assay for Monocytes
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The expression of various markers was estimated by overnight staining of monocytes and macrophages with antibodies to CD68, CD86, CD206 (1:100, Abcam, Bristol, UK), CD11b, and CD163 (1:100, Santa Cruz, Dallas, TX, USA). Thereafter, cells were washed thrice and incubated with secondary antibodies conjugated with FITC (1:200, Abcam) for 1 h, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, Darmstadt, Germany) as described previously [45 (link)]. Immunostaining analysis was performed with a Leica DM 4000 B fluorescent microscope and LAS AF software (Leica Microsystems, Wetzlar, Germany).
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