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Our Abpromise guarantee covers the use of ab9475 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/100 - 1/750. Predicted molecular weight: 33 kDa.|
|IP||1/100 - 1/750.|
|IHC-P||1/20 - 1/50.|
|IHC-Fr||1/20 - 1/50.|
|ICC/IF||Use at an assay dependent dilution. PubMed: 21825174|
|Flow Cyt||Use at an assay dependent dilution.
ab170191-Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.
Immunohistochemical analysis of paraffin embedded human tonsil tissue with ab9475 at 1 in 50 dilution.
Human peripheral blood lymphocytes stained with ab9475 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab9475, 0.1μg/1x106 cells) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.
ab9475 - immunohistochemistry
Formalin fixed paraffin embedded human tonsil stained with CD20, B cell using ABC and Fast Red chromagen.
Image from Polyak MJ et al, J Biol Chem 283:18545-52 (2008), Fig 5.CD20 associates with phosphorylated proteins after BCR stimulation. Ramos cells were unstimulated or stimulated with F(ab')2 anti-Igµ for the times and temperatures indicated. Cells were lysed in digitonin, and CD20 was Immunoprecipitated from cleared lysates. Immunoprecipitates were probed using anti-phosphotyrosine (ptyr) (n = 4). The membranes were stripped and reprobed for CD20 using ab9475 (lower panel).