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Thioglycollate stimulated peritoneal macrophages from C57/BL mice
Please note we cannot guarantee IHC-P, however should you use this application for this product then please use a specific protocol which can be found in the protocols section of our datasheet here.
Our Abpromise guarantee covers the use of ab6640 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 10 µg/ml. Use under non reducing condition. Detects a band of approximately 160 kDa (predicted molecular weight: 102 kDa). (predicted molecular weight of precursor protein: 102 kDa; protein is heavily glycosylated). Block in 5% milk for 1 hour.|
|IP||Use at an assay dependent concentration.|
|RIA||Use at an assay dependent concentration.|
|IHC-Fr||Use a concentration of 10 µg/ml.|
|Flow Cyt||1/10 - 1/50.
ab18536 - Rat monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
|ICC||Use at an assay dependent concentration.|
|IHC-FoFr||Use a concentration of 10 µg/ml.|
|ICC/IF||Use a concentration of 5 - 10 µg/ml.|
|IHC-R||Use a concentration of 10 µg/ml.|
ab6640 stained Raw 264.7 cells. The cells were 100% methanol fixed for 5 minutes at room temperatureand then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6640 at 5µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was ab150165 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed used at a 1/1000 dilution for 1 hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
This image is courtesy of an anonymous Abreview
ab6640 staining F4/80 in mouse spleen tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with paraformaldehyde and blocked with 5% serum for 45 minutes at 21°C. The sample was incubated with primary antibody (1/100 in PBS + 5% FBS) at 4°C for 16 hours. An Alexa Fluor® 488-conjugated goat anti-rat IgG (H+L) polyclonal (1/200) was used as the secondary antibody.
Flow cytometry analysis of J774 cells labelling F4/80 (red) with ab6640 at a dilution of 1/10 followed by streptavidin FITC secondary at 1/100 dilution. The blue line shows J774 cells stained with Rat anti mouse isotype control.
Flow cytometry analysis of peritoneal macrophages labelling F4/80 (red) with ab6640 at a dilution of 1/10. The blue line shows J774 cells stained with Rat anti mouse isotype control.
Flow cytometry analysis of J774 cells labelling F4/80 (red) with ab6640 at a dilution of 1/50 followed by goat anti rat IgG FITC secondary antibody at 1/100 dilution. The blue line shows J774 cells stained with Rat anti mouse isotype control.
ab6640 staining F4/80 (red) in Mouse intestine tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with acetone and blocked with 10% serum for 1 hour at 21°C. Samples were incubated with primary antibody (1/10µg/ml in PBS + 10% serum) for 16 hours at 4°C. An Alexa Fluor® 555-conjugated Donkey anti-rat IgG polyclonal (1/1000) was used as the secondary antibody. Blue - nuclei.