Frozen heart sections were fixed in 4°C acetone for 10 min and permeabilized with 0.3% Triton X-100. Then, cells was washed three times with PBS and treated with the first antibody at 37°C; 2 h later, cells were washed three times with PBS and treated with fluorescent-labeled secondary antibody for 1 h at 37°C. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). We continued with several rounds of washing and finished with mounting the coverslip onto a microscope slide using an anti-fade mounting medium. All of the immunofluorescence staining was photographed under a confocal microscope. The antibodies used were as follows: CD68 antibody (diluted at 1:200; Abcam), iNOS antibody (diluted at 1:100; Abcam), arginase antibody (diluted at 1:1,000; Abcam), and goat anti-mouse IgG (ab150115; Abcam).
Cd68 antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The CD68 antibody is a laboratory reagent used to detect the presence of the CD68 protein, which is a glycoprotein expressed on the surface of macrophages and monocytes. It is commonly used in immunohistochemistry and flow cytometry applications to identify and quantify these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Arginase antibody, by Abcam (2 mentions)
Inos antibody, by Abcam (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
Goat anti mouse igg ab150115, by Abcam (1 mentions)
Tripure reagent, by BioTeke (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Solarbio (1 mentions)
Tcs sp8 system, by Leica (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)
Cd163 antibody, by Abcam (1 mentions)
Monoclonal cd 11b antibody, by Santa Cruz Biotechnology (1 mentions)
Ventana immunostainer, by Roche (1 mentions)
Biotinylated griffonia, by Vector Laboratories (1 mentions)
Formalin free zinc fixative, by BD (1 mentions)
A1 inverted confocal microscope, by Nikon (1 mentions)
Fluoroshield mounting medium with dapi, by Abcam (1 mentions)
E cadherin antibody, by BD (1 mentions)
17 protocols using cd68 antibody
1
Immunofluorescence Staining of Frozen Tissues
2
Bovine Collagen-Induced Osteoarthritis Model
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Bovine type II collagen (CII) was purchased from Source Leaf Biological Technology Co., Ltd. (Shanghai, China). Hemotoxylin and SYBR Green were from Solarbio Life Sciences (Beijing, China),
and Eosin was obtained from Sangon Biotechnology (Shanghai, China). AKAP12, MMP-1 and CD206 antibodies were purchased from Proteintech, MMP-3 and MMP-13 antibodies were from Affinity, and
Goat anti-rabbit IgG was from ThermoFisher (Shanghai, China). CD68 antibody was obtained from Abcam (UK) and anti-CCR7 was from ABclonal (Wuhan, China). 3,3′-diaminobenzidine (DAB)
chromogenic solution was obtained from Fuzhou Maixin Biotech (Fuzhou, China). The primers used were synthesized by GenScript (Nanjing, China). TRIpure reagent was bought from BioTeke
(Beijing, China). Electrochemiluminescence (ECL) reagent was provided by Beyotime Biotechnology (Shanghai, China). Anti-collagen type II (CII) antibody ELISA kit was from FineTest (Wuhan,
China). The kits for detecting IL-1β, IL-6, tumor necrosis factor (TNF)-α and IL-10 levels were acquired from MultiSciences (Hangzhou, China).
and Eosin was obtained from Sangon Biotechnology (Shanghai, China). AKAP12, MMP-1 and CD206 antibodies were purchased from Proteintech, MMP-3 and MMP-13 antibodies were from Affinity, and
Goat anti-rabbit IgG was from ThermoFisher (Shanghai, China). CD68 antibody was obtained from Abcam (UK) and anti-CCR7 was from ABclonal (Wuhan, China). 3,3′-diaminobenzidine (DAB)
chromogenic solution was obtained from Fuzhou Maixin Biotech (Fuzhou, China). The primers used were synthesized by GenScript (Nanjing, China). TRIpure reagent was bought from BioTeke
(Beijing, China). Electrochemiluminescence (ECL) reagent was provided by Beyotime Biotechnology (Shanghai, China). Anti-collagen type II (CII) antibody ELISA kit was from FineTest (Wuhan,
China). The kits for detecting IL-1β, IL-6, tumor necrosis factor (TNF)-α and IL-10 levels were acquired from MultiSciences (Hangzhou, China).
3
Immunofluorescence Analysis of STAT6, CD11b, CD68, and CD163
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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).
4
Immunohistochemical Analysis of Inflammation Cells
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Tissues were fixed in 10% neutral-buffered formaldehyde for 2 h, transferred into 70% ethanol, and processed the next day by standard techniques. Immunohistochemistry for inflammation cells was performed on paraffin-embedded sections (4 µm) of tumors. Briefly, the paraffin sections were baked in an oven at 56 °C for 30 min. The sides were deparaffinized and rehydrated in xylene, followed by graded ethanol washes at room temperature. Antigen retrieval was achieved by boiling the slides in a microwave oven with 0.01 M, pH 6.0 sodium citrate buffer. Sides were then incubated in hydrogen peroxide (3% H2O2 in PBS) for 10 min at room temperature, followed by incubation in 5% fetal bovine serum/PBS for 1 h. The inflammation cells were stained with polyclonal CD 68 antibody (Abcam, Cambridge, MA, USA) and monoclonal CD 11b antibody (Santa Cruz, Dallas, TX, USA).
5
Histological Analysis of Liver and Adipose Tissue
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Liver and eWAT samples were fixed in 10% formalin. Hematoxylin and eosin staining was performed on 5‐μm sections from the paraffin‐embedded tissue blocks for conventional light microscopy analysis. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling staining of eWAT samples were deparaffinized and stained using the standard protocol. For immunohistochemical staining, liver sections were deparaffinized and immunostained with rabbit anti‐mouse clusters of differentiation (CD)68 antibody (Abcam) or rabbit monoclonal anti‐mouse CD3 antibody (Ventana Medical Systems, Inc.), using the automated Ventana immunostainer according to the manufacturer's recommendation.14 , 17 , 18
6
Quantification of Pancreatic Islet Cell
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Excised tissues were placed in formalin-free zinc fixative (BD Pharmingen) for 24–48 h and embedded in paraffin. CD3 antibody (BD Pharmingen), CD68 antibody (Abcam), insulin antibody (Abcam), and biotinylated Griffonia (Bandeiraea) simplicifolia lectin I (Vector Laboratories B1105) was used to stain zinc-fixed, paraffin-embedded tissue. Slides were incubated with CD3 antibody at a 1:100 overnight at 4°C, CD68 antibody at a 1:100 for 1 h at room temperature, insulin antibody at a 1:500 overnight at 4°C, or biotinylated lectin at a 1:5,000 overnight at 4°C. For capillary density quantification, lectin staining intensity was quantified as a percentage of islet area as previously described (22 (link)). For β-cell mass measurements, a minimum of four sections separated by at least 200 μm were chosen, stained for insulin, and then scanned with Aperio Scan Scope OS. Images were analyzed using Aperio Image Analysis v11.1.2.760 Toolkit algorithms Genie Classifier v1 and Color Deconvolution v9. Results were expressed as a percentage of insulin-positive area relative to the total area scanned. Total β-cell mass was calculated by normalizing β-cell percentage to total pancreas weight.
7
Immunohistochemical Characterization of Hepatic Organoids
8
Mitochondrial Staining and Immunofluorescence Analysis
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For mitochondrial staining, macrophages were incubated with MitoTracker® Red (Thermo Fisher, Waltham, MA, USA) at 37°C for 20 min. Then, cell slides or frozen tissue samples were fixed with 4% paraformaldehyde for 15 min at 4°C. After washing with PBS three times, cells were punched with 1% Triton and blocked with 2% horse serum for 1 hour at room temperature. Then, slides were incubated with primary antibody (LC3B antibody, 1 μg/ml; NLRP3 antibody, 1/200 dilution; and CD68 antibody, 1/100 dilution, all from Abcam) at 4°C overnight. After being washed with PBS three times, samples were incubated with the corresponding IgG-FITC or Rhodamine-conjugated secondary antibody (dilution factor 1 : 200) for 2 h at room temperature. Cell nuclei were counterstained by DAPI (1 mg/ml) for 5 min. After a final wash, the slides were visualized under a laser scanning confocal microscope (Olympus FV1200, Olympus, Tokyo, Japan).
9
Baicalin's Protective Effects in Inflammation
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Baicalin was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China), and it was resuspended using distilled water to 40 mg/mL for the animal experiments. Antibodies against P38, Bax, Bcl-2, Caspase-3, Erk1/2, Nrf2, HO-1 and β-action were purchased from Proteintech (Wuhan, China). JNK, p-JNK and all secondary antibodies used in Western blot were obtained from Cell Signaling Technology (Beverly, MA, USA). p-P38, p-Erk1/2, NQO-1, CD3 and MPO antibodies were from ABclonal Technology (Wuhan, China). CD68 antibody was provided by Abcam (Cambridge, MA). CD4 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Trizol, Hifair™ II 1st Strand cDNA Synthesis Super Mix and Hieff®qPCR SYBR®Green Master Mix were from Yeasen (Shanghai, China). The TUNEL apoptosis and immunohistochemistry kits were obtained from Wuhan Gugeshengwu Technology Co., Ltd. (Wuhan, China). All other regular reagents were obtained from Wuhan Gugeshengwu Technology Co., Ltd. unless otherwise specified.
10
Chloroquine and Bafilomycin A1 Autophagy Study
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Chloroquine was purchased from Sigma-Aldrich, and bafilomycin A1 (Baf A1) was from Selleck. The following antibodies in immunoblotting and immunohistochemistry were used: anti-LC3B, anti-cleaved caspase-3, anti-Bcl-2, anti-p-Akt, anti-Akt, anti-p-PI3K, anti-PI3K, anti-mTOR, anti-p-mTOR and anti-gD were obtained from Cell Signaling Technology (Beverly, MA, United States). The antibodies for CD3 and MPO were from ABclonal Technology (Wuhan, China). Bax antibody was purchased from Proteintech (Wuhan, China). CD68 antibody was provided by Abcam (Cambridge, MA). CD4 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody for β-actin was purchased from Servicebio Co.,Ltd. (Wuhan, China).
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