Western blot - Anti-CD4 antibody [EPR19514] (ab183685)

The expression profile observed is consistent with what has been described in the literature (PMID: 2155425).

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1:3 seconds; Lanes 2,3,4,5 and 6:30 seconds

All lanes:

Western blot - Anti-CD4 antibody [EPR19514] (ab183685) at 1/2000 dilution

Lane 1:

Mouse thymus lysate at 20 µg

Lane 2:

Mouse liver tissue lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse kidney tissue lysate at 20 µg

Lane 5:

Raw264.7 (mouse abelson murine leukemia virus-induced tumor) whole cell lysate at 20 µg

Lane 6:

NIH/3T3 (mouse embryo) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 51 kDa

Observed band size: 51 kDa

Immunohistochemistry (Frozen sections) - Anti-CD4 antibody [EPR19514] (ab183685)

IHC image of CD4 staining in a section of frozen normal Mouse Spleen.

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 ab183685 (Rabbit monoclonal [EPR19514] to CD4) at 1/200 and ab243840 at 1μg/ml, to show the distinct staining of B cells and T cells. The section was then incubated with ab150165 (Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preabsorbed, (Shown in green) 1/1000) and ab150080 (Goat Anti-Rabbit IgG H&L (Alexa Fluor®594) (Shown in red) 1/1000) for 1 hour at room temperature. The secondary-only control insert image is taken from an identical assay without primary antibody. DAPI was used to stain the cell nuclei (blue). 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.

Immunohistochemistry (Frozen sections) - Anti-CD4 antibody [EPR19514] (ab183685)

Immunohistochemical analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized frozen Mouse spleen tissue labeling CD4 with ab183685 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

The result showed membrane staining on mouse spleen.

The nuclear counterstain is DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 at 1/1000 dilution.

Antigen retrieval: Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).