The following antibodies were used in the current study: STAT6 antibody, CD11b antibody, CD68 antibody, and CD163 antibody (all from Abcam, Cambridge, MA, USA). Tissue samples from STAT6−/− or WT mice were fixed with 4% paraformaldehyde for 12 h followed by 30% sucrose overnight. Texas Red-conjugated rabbit-specific secondary antibody and/or FITC-conjugated mouse-specific secondary antibody (Biolegend, London, UK) were used for immunofluorescence analysis. CD68 and CD163 fluorescence was observed, and images were captured with a Leica TCS SP8 system (Leica, Allendale, NJ, USA).
Cd163 antibody
Manufactured by Abcam
Sourced in United States
The CD163 antibody is a protein that specifically binds to the CD163 receptor, which is expressed on the surface of certain immune cells. It is commonly used in research applications to identify and study these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Inos antibody, by Abcam (2 mentions)
Tcs sp8 system, by Leica (1 mentions)
Cd68 antibody, by Abcam (1 mentions)
Cd11b antibody, by Abcam (1 mentions)
Texas red conjugated rabbit specific secondary antibody, by BioLegend (1 mentions)
Fitc conjugated mouse specific secondary antibody, by BioLegend (1 mentions)
Stat6 antibody, by Abcam (1 mentions)
F4 80, by Santa Cruz Biotechnology (1 mentions)
Cd206, by Proteintech (1 mentions)
F4 80, by Affinity Biosciences (1 mentions)
Eclipse c1, by Nikon (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Cd9 antibody, by Proteintech (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Abi 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Nupagenovex bis tris mini gels, by Thermo Fisher Scientific (1 mentions)
6 protocols using cd163 antibody
1
Immunofluorescence Analysis of STAT6, CD11b, CD68, and CD163
2
Profiling Macrophage Subsets in Decalcified Mandibular Samples
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The left hemimaxillas were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 2 months. The samples were embedded in paraffin and were sliced into serial paraffin sections (5 μm) in the mesiodistal direction on the mandibular first molars. The sections were sealed at room temperature in 10% serum and 1% BSA for 2h. Subsequently, they were incubated with CD163 antibody (1:200) (Abcam, Cambridge, MA, USA) or iNOS antibody (1:100) (Abcam, Cambridge, MA, USA) at 4°C overnight, and then incubated with secondary antibody (1:20,000) (Abcam, Cambridge, MA, USA) at room temperature for 1h. After the DAB reaction, nuclei were counterstained with hematoxylin for 5 min. Finally, images were captured by microscope camera at 400× magnification. The positive cell counting was conducted by two examiners.
For the immunofluorescence assay, sections were incubated with (1:100) F4/80 (Affinity, Jiangsu, China) and iNOS (1:100) (Proteintech, Wuhan, China) or F4/80 (1:100) (Santa Cruz, Santa Cruz, California, USA) and CD206 (1:200) (Proteintech, Wuhan, China) at 4°C overnight, and then incubated with secondary antibody (1:20,000) (Abcam, Cambridge, MA, USA) for 1h. Cell nuclei were counterstained with DAPI. Samples were observed under a fluorescence microscope and analyzed using NIH IMAGEJ analysis software.
For the immunofluorescence assay, sections were incubated with (1:100) F4/80 (Affinity, Jiangsu, China) and iNOS (1:100) (Proteintech, Wuhan, China) or F4/80 (1:100) (Santa Cruz, Santa Cruz, California, USA) and CD206 (1:200) (Proteintech, Wuhan, China) at 4°C overnight, and then incubated with secondary antibody (1:20,000) (Abcam, Cambridge, MA, USA) for 1h. Cell nuclei were counterstained with DAPI. Samples were observed under a fluorescence microscope and analyzed using NIH IMAGEJ analysis software.
3
Immunohistochemical Analysis of CD163 in ccRCC
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The ccRCC and normal renal tissue were immobilized in 10% phosphate formalin solution, then paraffin embedded, and sectioned to 5 μm thickness. The paraffin sections were then dewaxed and antigen repaired. Next, the sections were closed with goat serum at room temperature for 30 min and incubated overnight with CD163 antibody (1 : 200) at 4°C (Abcam, Cambridge, MA, USA). The sections were then incubated in CY3-labeled fluorescent secondary antibody for 1 h and DAPI dye for 10 min. Finally, the images were observed and collected under a fluorescence microscope (Nikon Eclipse C1, Nikon, Tokyo, Japan).
4
Immunofluorescence Imaging of CD163 and PD-L1
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The slides were baked at 60 °C for 30 min, deparaffinized with xylene and dehydrated with ethanol. Antigen retrieval in Tris-EDTA buffer (pH = 9.0) at high pressure for 5 min, followed by cooling down at room temperature naturally. Nonspecific antigen was blocked by 5% BSA, all slides were then incubated with rabbit polyclonal CD163 antibody (Abcam, USA) and mouse monoclonal PD-L1 antibody (Abcam, USA) overnight at 4 °C. After rinsing with PBST, the slices were incubated with fluorescent secondary antibody at 37 °C for 1 h. The slides were counterstained with DAPI at room temperature for 15 min, then analysed using a ZEISS LSM 710 confocal microscope (Germany).
5
Immunohistochemical Analysis of Tumor Markers
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Tumors were fixed, embedded in paraffin and sectioned at a thickness of 4 μm. After deparaffinization and rehydration, sections were blocked with 3% H2O2 and incubated with iNOS antibody (1:100) (Abcam, MA, USA), TNF-a antibody (1:200) (Abcam, MA, USA), CD163 antibody (1:200) (Abcam, MA, USA), CD9 antibody (1:100) (Proteintech, MA, USA), PPARδ antibody (1:200) (Proteintech, MA, USA) overnight at 4°C, followed by incubation in secondary biotinylated antibody for 30 min at 37°C. Blots were visualized with DAB solution and counterstained with haematoxylin. Pictures were taken under a light microscope. Stained slides were reviewed and scored independently by two observers blinded to the samples' information. Scores were determined by a combination of the proportion of positively stained cells and the intensity of staining as follows: 0 (no positive cells), 1 (< 10% positive cells), 2 (10-50% positive cells), and 3 (> 50% positive cells).
Staining intensity was classified according to the following criteria: 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellow brown), and 3 (strong staining = brown). Staining index was calculated as the staining intensity score ⅹ the proportion score. Using this method, IHC data was evaluated by determining the staining index with scores ranging from 0, 1, 2, 3, to 9.
Staining intensity was classified according to the following criteria: 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellow brown), and 3 (strong staining = brown). Staining index was calculated as the staining intensity score ⅹ the proportion score. Using this method, IHC data was evaluated by determining the staining index with scores ranging from 0, 1, 2, 3, to 9.
6
Quantitative Analysis of CD163 Expression
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Total RNA (500 ng) was extracted using Trizol DNase-treated, and cDNA synthesized using oligo-dT primers and SuperScriptIII (Life Technologies). Quantitative real-time PCR reactions were performed in triplicate using an ABI 7300 Real-Time PCR System and SYBR Green PCR Master Mix (Life Technologies). The comparative C T method was used for the comparative quantification of gene expression, with results from each time point normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and expressed as the fold difference over that in human brain (Clontech, Takara Bio, Otsu, Japan). Primer sequences were those reported previously. [32] [33] [34] [35] Western Blotting Equal amounts of each protein lysate were separated on 4-12% NuPAGE Novex Bis-Tris Mini Gels (Life Technologies). Blots were incubated with a CD163 antibody (EPR14643; Abcam). Alkaline phosphatase secondary antibodies were detected using the WesternBreeze Chemiluminescent Kit detection system according to the manufacturer's instructions (Life Technologies), and images were captured on X-ray film.
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