CBA mouse spleen cells and thymocytes
ab22378 is not expected to work in standard western blots, under denaturing conditions, or in IHC on formalin-fixed tissue. To our knowledge, ab22378 will only stain frozen sections or fresh cells, and will only immunoprecipitate the antigen from cell lysates in mild detergents such as NP40 or Triton-X100.
Our Abpromise guarantee covers the use of ab22378 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.|
|Flow Cyt||Use 0.1-1µg for 106 cells.
ab18536 - Rat monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
|IHC-Fr||Use at an assay dependent concentration.|
ab22378 staining CD8 in murine bone marrow cells by ICC/IF (immunocytochemistry/immunofluorescence). Cells were formaldehyde fixed prior to blocking in 2% BSA for 30 minutes at 20°C. The primary antibody (1/200) was incubated with the sample for 9 hours at 4°C. An undiluted Alexa Fluor® 555-conjugated goat anti-rat polyclonal was used as the secondary. The nuclei were stained with DAPI.
ab22378 staining CD8 in mouse spleen tissue sections by IHC-Fr (Frozen sections). Tissue samples were fixed with acetone and blocked with 5% serum for 2 hours at 25°C. The sample was incubated with primary antibody (1/250 in PBS-Tween) at 4°C for 12 hours. An Alexa Fluor® 594-conjugated Goat polyclonal to rat IgG (1/250) was used as secondary antibody. Nuclei were stained with DAPI.
Overlay histogram showing mouse spleen cells (C57BL/6) stained with ab22378 (red line). The cells were incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab22378, 0.1μg/1x10^6 cells) for 30 min at 4ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rat IgG (H&L) (ab150157) at 1/2000 dilution for 30 min at 4ºC. Isotype control antibody (black line) was rat IgG2b [RTK4530] (ab18541, 2μg/1x10^6 cells) used under the same conditions. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
Normal mouse (CBA) spleen cells (red cell lysed) double stained with ab22378 (0.3 µg/mL, detected with Alexa Fluor® 488-conjugated monoclonal mouse anti-rat antibody) and an APC conjugated hamster anti-mouse CD3 antibody. ab22378 stains an expected subpopulation of CD8 alpha positive T cells.
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