Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling CD4 with ab237722 at 1/4000 dilution, followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse spleen. The section was incubated with ab237722 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling CD4 with ab237722 at 1/4000 dilution, followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat spleen. The section was incubated with ab237722 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

All lanes : Anti-CD4 antibody [CAL4] (ab237722) at 1/1000 dilution

Lane 1 : Mouse thymus lysate
Lane 2 : Mouse spleen lysate
Lane 3 : Mouse lymph node lysate
Lane 4 : Rat thymus lysate
Lane 5 : Rat spleen lysate
Lane 6 : Rat lymph node lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 51 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse spleen tissue labeling CD4 with ab237722 at 1/100 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Positive staining on T cell surface (PMID: 26994219) is observed. Counterstained with DAPI (blue). Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution. Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween-20 permeabilized mouse splenocytes labeling CD4 with ab237722 at 1/40 (right panel) compared with a Rabbit monoclonal IgG (ab172730) (left panel). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody. Cells were surface stained with anti-CD4 conjugated to Alexa Fluor^® 647. Then fixed with 2% PFA for 10min and intracellularly stained with rabbit IgG (Left) or ab237722 (Right). They recognize the same cell population.

CD4 was immunoprecipitated from 0.35 mg mouse thymus lysate with ab237722 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab237722 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/5000 dilution.
Lane 1: Mouse thymus lysate 10 µg (Input).
Lane 2: ab237722 IP in mouse thymus lysate .
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab237722 in mouse thymus lysate .
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 30 seconds.

102kDa band represents CD4 dimer based on literature. (PMID: 20739067).

Formalin-fixed, paraffin-embedded mouse spleen tissue stained for CD4 using ab237722 at 0.5 µg/ml in immunohistochemical analysis.