Cryostat sections (4μm) of frozen kidneys were fixed with ice-cold acetone for 10min, and blocked with 10% goat serum to decrease background staining. FITC-conjugated goat anti-mouse C3 Ab (4.0 mg/ml, MP Biomedicals) was used directly at 1:500 dilution. Under ×200 magnification, 10 viewing fields from sections of each animal were photographed. Areas of positive staining were highlighted and the fluorescence intensity of C3 was determined using the plugin “Measure particles” of ImageJ software. F4/80-positive mononuclear phagocytes were visualized by staining with a rat anti-mouse F4/80 Ab (1.0 mg/ml, AbD seroTEC) used at 1:50 dilution followed by Alexa Fluor 555-goat anti-rat IgG (2.0 mg/ml, Invitrogen) used at 1:2000 dilution. Under 400x magnification, F4/80+ cells were counted by examining ten viewing fields randomly selected from the outer medulla on each slide.
Rat anti mouse f4 80 ab
Manufactured by Bio-Rad
The Rat anti-mouse F4/80 Ab is a monoclonal antibody that specifically binds to the F4/80 antigen, a glycoprotein expressed on the surface of murine macrophages. This antibody can be used to identify and isolate macrophage populations in mouse tissues and cell samples.
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5 protocols using rat anti mouse f4 80 ab
1
Quantifying Kidney Complement C3 and Macrophages
2
Quantifying Kidney Complement C3 and Macrophages
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Cryostat sections (4μm) of frozen kidneys were fixed with ice-cold acetone for 10min, and blocked with 10% goat serum to decrease background staining. FITC-conjugated goat anti-mouse C3 Ab (4.0 mg/ml, MP Biomedicals) was used directly at 1:500 dilution. Under ×200 magnification, 10 viewing fields from sections of each animal were photographed. Areas of positive staining were highlighted and the fluorescence intensity of C3 was determined using the plugin “Measure particles” of ImageJ software. F4/80-positive mononuclear phagocytes were visualized by staining with a rat anti-mouse F4/80 Ab (1.0 mg/ml, AbD seroTEC) used at 1:50 dilution followed by Alexa Fluor 555-goat anti-rat IgG (2.0 mg/ml, Invitrogen) used at 1:2000 dilution. Under 400x magnification, F4/80+ cells were counted by examining ten viewing fields randomly selected from the outer medulla on each slide.
3
Isolation of Murine Peritoneal and Bone Marrow Macrophages
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Pyrogen-free, 8- to 10-wk-old C57BL/6 mice were purchased from Harlan (Oxon, U.K.). Mice were housed in barrier cages under controlled environmental conditions (12/12 hrs light/dark cycle, 55 ± 5% humidity, 23 °C) in the University Biological Services Unit, University College Cork. All animal procedures were performed in the University Biological Services Unit under a license from the Department of Health (Republic of Ireland) and with ethical approval granted from the University College Cork Ethics Committee. The methods applied in this study were carried out in accordance with the approved guidelines.
Peritoneal macrophages were collected from C57BL/6 mice by peritoneal lavage and incubated with DMEM containing 10% heat-inactivated FCS for 90 min to remove non-adherent cells, as described previously46 (link). BMMs were isolated from the femurs and tibias of C57BL/6 mice and cultured in DMEM containing 20% heat-inactivated FCS, penicillin (100 units/ml), streptomycin sulfate (100 μg/ml), and supplemented with 10 ng/ml of recombinant mouse macrophage-CSF (R&D Systems, Minneapolis, MN, USA) for 7 days at 37 °C in a humidified 5% CO2 atmosphere, as described previously46 (link). The purity of both peritoneal macrophages and BMMs was >95%, as confirmed by FAC Scan analysis of the positive F4/80 Ag staining with a rat anti-mouse F4/80 Ab (Serotec, Oxford, U.K.).
Peritoneal macrophages were collected from C57BL/6 mice by peritoneal lavage and incubated with DMEM containing 10% heat-inactivated FCS for 90 min to remove non-adherent cells, as described previously46 (link). BMMs were isolated from the femurs and tibias of C57BL/6 mice and cultured in DMEM containing 20% heat-inactivated FCS, penicillin (100 units/ml), streptomycin sulfate (100 μg/ml), and supplemented with 10 ng/ml of recombinant mouse macrophage-CSF (R&D Systems, Minneapolis, MN, USA) for 7 days at 37 °C in a humidified 5% CO2 atmosphere, as described previously46 (link). The purity of both peritoneal macrophages and BMMs was >95%, as confirmed by FAC Scan analysis of the positive F4/80 Ag staining with a rat anti-mouse F4/80 Ab (Serotec, Oxford, U.K.).
4
Histological Analysis of Inflammatory Joint Changes
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At day 10, both forepaws and the entire right hind limb, including the paw, ankle, and knee were surgically removed and fixed immediately in 10% buffered formalin (Biochemical Sciences). The preparation of knee joint samples and histological analysis using toluidine blue (T blue) dye were performed as previously described (14 (link), 15 (link)). All sections were read by a trained observer who was also blinded to the treatment and to the disease activity score of each mouse. The joint sections from mice were scored for the changes in inflammation, pannus, cartilage damage, and bone damage on a scale of 0–5. C3 deposition in the joints (synovium and cartilage) was localized with polyclonal goat anti-mouse C3 antisera (ICN Pharmaceuticals) and scored as previously described (13 (link)). Macrophages infiltration in the knee joints were localized with rat anti-mouse F4/80 Ab (Bio-Rad) and scored using a scale from 0 to 4.
5
Histological Analysis of Inflammatory Joint Changes
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