The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
IHC-P: Ready-to-use for 10 min at RT. Perform heat mediated antigen retrieval by boiling tissue sections in 10mM citrate buffer, pH 6.0 for 10 min followed by cooling at RT for 20 min before commencing with IHC staining protocol.
Not yet tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
Identifies cytotoxic/suppressor T-cells that interact with MHC class I bearing targets. CD8 is thought to play a role in the process of T-cell mediated killing. CD8 alpha chains binds to class I MHC molecules alpha-3 domains.
Defects in CD8A are a cause of familial CD8 deficiency (CD8 deficiency) [MIM:608957]. Familial CD8 deficiency is a novel autosomal recessive immunologic defect characterized by absence of CD8+ cells, leading to recurrent bacterial infections.
CD8 beta tissue specificity: Isoform 1, isoform 3, isoform 5, isoform 6, isoform 7 and isoform 8 are expressed in both thymus and peripheral CD8+ T-cells. Expression of isoform 1 is higher in thymus CD8+ T-cells than in peripheral CD8+ T-cells. Expression of isoform 6 is higher in peripheral CD8+ T-cells than in thymus CD8+ T-cells.
CD8 beta PTM: Phosphorylated as a consequence of T-cell activation.
Human peripheral blood lymphocytes stained with ab27605 (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 (ab27605, 1/4 dilution) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (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.
Ogembo JG et al. SIRPa/CD172a and FHOD1 are unique markers of littoral cells, a recently evolved major cell population of red pulp of human spleen. J Immunol188:4496-505 (2012).
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