Immunohistochemical analysis was performed on decalcified paraffin-embedded tissue sections as described previously [28 (link)]. The anti-F4/80 antibody clone CI:A3-1 (Abcam, Cambridge, MA, USA) that recognizes the mouse F4/80 antigen, a cell surface glycoprotein expressed at high levels on various murine macrophages, was used to detect macrophages in arthritic joints. Detection of the primary antibody was performed using the VECTASTAIN® Elite ABC kit (Vector Laboratories, Burlingame, CA, USA), followed by 3,3-diaminobenzidine (Vector Laboratories) incubation and nuclear staining with hematoxylin. Six randomly chosen fields per slide were analyzed and averaged.
Ci a3 1
Manufactured by Abcam
Sourced in United States, United Kingdom
The CI:A3-1 is a laboratory equipment product. It is a core component designed to perform a specific function within the laboratory setting. No further details about the intended use or functionality of this product can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Anti dykddddk, by BioLegend (1 mentions)
Clone m1 70, by BD (1 mentions)
Il 4 il 13, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
F4 80, by Abcam (1 mentions)
Cy3 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Prolong gold antifade mountant with 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Cellsens software, by Olympus (1 mentions)
Blocking one histo, by Nacalai Tesque (1 mentions)
Ab28364, by Abcam (1 mentions)
3 3 diaminobenzidine tablets, by Merck Group (1 mentions)
5 protocols using ci a3 1
1
Macrophage Detection in Arthritic Joints
2
Immunophenotyping of Mouse Myeloid Cells
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Cells (5 × 105) were placed into polypropylene tubes and pelleted by centrifugation at 200 × g for 5 mins at 4 °C. These were then resuspended in FACS buffer (PBS containing 2% FCS, 25 mM HEPES and 5 mM EDTA) supplemented with mouse IgG and mouse SeroBlock FcR® (AbD Serotec, Oxford, UK) and left on ice for 30 min. For studies using CHO cells, the cell pellet was resuspended in FACS buffer alone. Specific staining was conducted using the following antibodies and appropriate isotype controls: F4/80 (1:100, FITC, clone CI:A3-1, Abcam, Cambridge, UK), Ly-6B.2 (1:100, Alexa Fluor® 647, clone 7/4, Abd Serotec), Ly-6G (1:100, PE, clone 1A8, BD Biosciences, Oxford, UK), CD11b (1:100, FITC, clone M1/70, BD Biosciences) anti-HA (1:50, APC, clone GG8-1F3.3.1, Miltenyi Biotec, Surrey, UK), anti-c-myc (1:10, FITC, clone SH1-26E7.1.3, Miltenyi Biotec), anti-FLAG (anti-DYKDDDDK, 1:50, PE, clone L5, Biolegend, London, UK) for 1 hour on ice. Cells were then pelleted by centrifugation at 200 × g for 5 mins at 4 °C, resuspended in 1% formaldehyde and analysed using a Dako Cyan ADP flow cytometer (Beckman Coulter Ltd, High Wycombe, UK) and FlowJo software (V10, Tree Star, Ashland, USA).
3
Mouse BMDM Isolation and Polarization
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Mouse BMDMs were prepared from healthy male mice (8–12 weeks old C57BL/6) as described34 (link). In brief, the whole bone marrow was derived and cultured in culture dish (Corning), whereas the cells were induced to macrophage phenotype using DMEM medium containing 10% FBS, 1% Penicillin–Streptomycin solution and 15% L929 conditioned medium with MCSF for 7 days at 37 °C and 5% CO2. This process routinely yielded a macrophage population of > 95% purity as assessed by flow cytometry for CD11b (eBioscience, M1/70, 1:100) and F4/80 (abcam, CI:A3-1, 1:400). The additional M1 induction was achieved by stimulating macrophages with LPS (50 ng ml−1) (L3129 Sigma) and the M2 induction was achieved by inducing BMDMs with IL-4/IL-13 (20 ng ml−1) (PeproTech) for 3 days.
4
Quantifying Allograft Immune Response
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LP was visualized by anti-PEG primary antibody (PEG-B-47b; Abcam, United Kingdom) on 4-µm frozen sections. Fat area % was determined on hematoxylin and eosin-stained 4-µm paraffin brown adipose tissue sections. Banff classification was performed by a trained nephropathologist according to the actual consensus on 2 µm periodic acid Schiff stained paraffin sections of the allograft.26 (link) Immunohistochemical stainings with CD3 and F4/80 antibodies (SP7 and CI:A3-1; both from Abcam) were performed on 2 µm paraffin sections and visualized using Cy3-conjugated secondary antibodies (Jackson ImmunoResearch). Sections were sealed with ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific). Semiquantitative scoring of the CD3+ T-cell and F4/80+ macrophage infiltrates was performed at 200-fold magnification on a Leica imaging microscope and scored on a 0–4 scale; 0 = no cells; 1= <2; 2 = 3–10; 3 = 11–25; 4 = >25 cells and dense cell accumulation.
5
Histological Analysis of Wound Healing
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For histological analysis, mice were killed 3, 7 and 14 days after treatment. The harvested wound tissues were fixed in 4% paraformaldehyde for paraffin embedding. Sections (6 μm thick) were used for various staining techniques: hematoxylin-eosin (HE) staining, Masson’s trichrome staining, Picrosirius red staining26 (link), and immunohistochemistry (IHC) staining for CD31 (ab28364, Abcam) and F4/80 (CI:A3-1, Abcam). For IHC, sections were deparaffinized, incubated for 20 min with 3% hydrogen peroxide to block endogenous peroxidase activity, incubated with Blocking One Histo (Nacalai Tesque) at room temperature, and then incubated with primary antibodies at 4 °C overnight. All sections were visualized using Histofine Simplestaining mouseMAX-PO (rabbit or rat) (Nichirei Biosciences, Inc.) and 3,3′-Diaminobenzidine tablets (Sigma-Aldrich). Stained sections were viewed by microscopy (Olympus IX83, Olympus), and digital images were acquired with a DP80 camera using the cellSens software (Olympus). Picrosirius red stained sections were observed using a polarized microscope. HE-stained sections were used to evaluate the re-epithelialization and granulation of wounds. Images were optimized globally and assembled into figures using Adobe Photoshop.
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