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Tissue/ cell preparation (Human). (Leucocytes of a patient suffering from a LGL-type leukaemia).
Abcam is committed to meeting high standards of ethical manufacturing and as such, we will be discontinuing this product, which has been generated by the ascites method, within the next year. We are sorry for any inconvenience this may cause. We would recommend antibody ab8220 as a replacement.
Our Abpromise guarantee covers the use of ab657 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use at an assay dependent concentration. PubMed: 24991932|
|IHC-P||Use at an assay dependent concentration.|
|Flow Cyt||Use 0.1-1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.|
|IHC-Fr||Use a concentration of 10 µg/ml.|
|IP||Use at an assay dependent concentration.|
|WB||Use a concentration of 2 µg/ml. Use under non reducing condition.
BSA is recommended for blocking.
|Functional Studies||Use at an assay dependent concentration. The antibody induces high-affinity conformation of LFA-1 complex.|
Human peripheral blood lymphocytes stained with ab657 (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 22ºC. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37ºC. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4ºC. Cells were then incubated with the antibody (ab657, 1μg/1x106 cells) for 30 min at 4ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 4ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 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.
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