Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CTLA4 with ab237712 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on human tonsil is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins.

The section was incubated with ab237712 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

All lanes : Anti-CTLA4 antibody [CAL49] (ab237712) at 1/1000 dilution

Lane 1 : Untreated human PBMC (human peripheral blood mononuclear cell) whole cell lysate
Lane 2 : Human PBMC (treated with 10µg/ml PHA for 2 days) whole cell lysate
Lane 3 : Untreated mouse splenocyte whole cell lysate
Lane 4 : Mouse splenocyte (treated with 2.5µg/ml Concanavalin A (ConA) for 3 days) whole cell 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: 25 kDa
Observed band size: 25 kDa


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween 20 permeabilized mouse splenocytes (treated with 2.5μg/ml Concanavalin A (ConA) for 3 days) cells labeling CTLA4 with ab237712 at 1/400 (Right) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Left).

Cells were surface stained with anti-CD3 conjugated to Alexa Fluor^® 647. Then fixed with 2% PFA for 10min followed by intracellular staining with rabbit IgG (Left) and ab237712 (Right).

Goat Anti-Rabbit IgG Fc (Alexa Fluor^® 488) preadsorbed (ab150097), at 1/2000 dilution was used as the secondary antibody.

CTLA4 was immunoprecipitated from 0.35 mh human tonsil lysate with ab237712 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab237712 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/1000 dilution.

Lane 1: Human tonsil lysate 10 μg (Input).
Lane 2: ab237712 IP in human tonsil lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab237712 in human tonsil lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.

Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween 20 permeabilized human PBMC (peripheral blood mononuclear cell) (treated with 10μg/ml PHA for 2 days) cells labeling CTLA4 with ab237712 at 1/400 (Right) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Left).

Cells were surface stained with anti-CD3 conjugated to Alexa Fluor^® 647. Then fixed with 2% PFA for 10min followed by intracellular staining with rabbit IgG (Left) and ab237712 (Right).

Goat Anti-Rabbit IgG Fc (Alexa Fluor^® 488) preadsorbed (ab150097), at 1/2000 dilution was used as the secondary antibody.

Formalin-fixed, paraffin-embedded human breast carcinoma tissue stained for CTLA4 using ab237712 at 0.25 μg/ml in immunohistochemical analysis.

Incubate with primary antibody for 75 minutes at room temperature.

Formalin-fixed, paraffin-embedded human lymph node tissue stained for CTLA4 using ab237712 at 0.25 μg/ml in immunohistochemical analysis.

Incubate with primary antibody for 75 minutes at room temperature.