Anti-CTLA4 antibody [CAL49]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTLA4 antibody [CAL49] (AB237712)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling CTLA4 with ab237712 at a concentration of 1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab237712 Anti-CTLA4 antibody [CAL49] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control. Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CTLA4 antibody [CAL49] (AB237712)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) (top) or PBMCs treated with 5µg/ml PHA for 48 Hours with ab237712 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction. Cells were then fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min followed by the antibody ab237712 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 0.2 μg/ml (1/10,300)) for 30 min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/2000 for 30min at 22°C.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTLA4 antibody [CAL49] (AB237712)

Tissue Microarrays stained for "Anti-CTLA4 antibody [CAL49]" using "ab237712"in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0) for 30 minutes. The sections were incubated with ab237712 for 30 mins at room temperature followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTLA4 antibody [CAL49] (AB237712)

Immunohistochemical analysis of paraffin-embeded Rhesus monkey tonsil tissue labeling CTLA4 with ab251599 followed by Polink 1 Polymer HRP anti-Rabbit IgG.

Heat mediated antigen retrieval-Buffer/Enzyme Used : Dako pH9.

This data was developed using the same antibody clone in a different buffer formulation (ab251599).

This image is courtesy of Dr. Chi Ngai Chan

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTLA4 antibody [CAL49] (AB237712)

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CTLA4 antibody [CAL49] (AB237712)

Intracellular flow cytometric analysis of2% 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTLA4 antibody [CAL49] (AB237712)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CTLA4 antibody [CAL49] (AB237712)

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CTLA4 antibody [CAL49] (AB237712)

Intracellular 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.

• IP

Supplier Data

Immunoprecipitation - Anti-CTLA4 antibody [CAL49] (AB237712)

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.

All lanes:

Immunoprecipitation - Anti-CTLA4 antibody [CAL49] (ab237712)

Predicted band size: 24 kDa

Observed band size: 25 kDa

false

• WB

Lab

Western blot - Anti-CTLA4 antibody [CAL49] (AB237712)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CTLA4 antibody [CAL49] (ab237712) at 1/1000 dilution

Lane 1:

Untreated human PBMC (human peripheral blood mononuclear cell) whole cell lysate at 20 µg

Lane 2:

Human PBMC (treated with 10μg/ml PHA for 2 days) whole cell lysate at 20 µg

Lane 3:

Untreated mouse splenocyte whole cell lysate at 20 µg

Lane 4:

Mouse splenocyte (treated with 2.5μg/ml Concanavalin A (ConA) for 3 days) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 24 kDa

Observed band size: 25 kDa

false

Exposure time: 3min