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Tissue/ cell preparation (Human). (Leucocytes of a patient suffering from a LGL-type leukaemia).
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.