Anti-MHC class I抗体[OX18] (ab6405)

Lewis rat splenocytes stained with ab6405 (right) or mouse IgG1 kappa; (ab170190) isotype (left). Cells were incubated for 30 min on ice in 1x PBS containing 10 % rat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab6405) or mouse IgG1 kappa; (ab170190) isotype (1x 10⁶ in 100 µL at 0.2 µg/mL) for 30 min on ice. The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150117) was used at 1/2000 dilution for 30 min on ice. The cells were simultaneously stained with CD3. Acquisition of >30,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. Events were gated on live single cells.

IHC image of MHC Class I staining in a section of frozen normal rat placenta*.

The section was fixed using 10% formaldehyde in 1XPBS for 10 minutes. No antigen retrieval step was performed prior to staining. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M glycine and 1% (w/v) BSA for 1h at room temperature.  The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab6405 (shown in green) at 1 μg/ml. The section was then incubated with ab150117 (Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 488, 1/1000)) presabsorbed for 1 hour at room temperature. The secondary-only control image is taken from an identical assay without primary antibody. The section was then mounted using Fluoromount^® .

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antibody concentrations and incubation times.

*Tissue obtained from Charles River.