Anti-CD40 antibody [EPR20540] - Low endotoxin, Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] - Low endotoxin, Azide free (AB223546)

Immunohistochemical analysis of paraffin-embedded human large B cell lymphoma tissue labeling CD40 with ab213205 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membranous staining on human large B cell lymphoma is observed [PMID : 7507299].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS BSA glycerol and sodium azide (ab213205).

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] - Low endotoxin, Azide free (AB223546)

This IHC data was generated using the same anti-CD40 antibody clone [EPR20540] in a different buffer formulation (cat# ab213205).

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD40 with ab213205 at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Membranous and cytoplasmic staining on germinal center of human tonsil is observed [PMID : 10360965] [PMID : 7507299] [PMID : 24452203].

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Mass Cytometry

Collaborator

Mass Cytometry - Anti-CD40 antibody [EPR20540] - Low endotoxin, Azide free (AB223546)

Imaging Mass Cytometry™ (IMC™) image of human tonsil tissue stained with Anti-CD40 antibody [EPR20540]. ab223546 (carrier-free antibody, purified) was metal-conjugated using a Maxpar® Antibody Labeling Kit from Fluidigm. Immunostaining was performed according to Fluidigm’s protocols. Briefly, slides were subject to deparaffinization and heat-induced epitope retrieval, followed by overnight incubation at 4°C with an antibody cocktail containing metal-tagged antibodies in blocking buffer. Slides were subsequently washed with 0.2% Triton-X and 1x PBS, counterstained with Cell-ID™ Intercalator-Ir diluted at 1/400 in 1x PBS for 30 min at room temperature, rinsed for 5 min with distilled H2O, and air-dried prior to IMC™ acquisition. IMC™ acquisition was performed using the Fluidigm Hyperion™ Imaging System. Imaging Mass Cytometry™, IMC™, Cell-ID™, Hyperion™ and Maxpar® are trademarks of Fluidigm Canada

This image is courtesy of the Single Cell & Imaging Mass Cytometry Analysis Platform, Goodman Cancer Research Centre, McGill University

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] - Low endotoxin, Azide free (AB223546)

Tissue Microarrays stained for "Anti-CD40 antibody [EPR20540]" using "ab213205"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 ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab213205 at +4°C overnight followed by Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500.

• WB

Lab

Western blot - Anti-CD40 antibody [EPR20540] - Low endotoxin, Azide free (AB223546)

This data was developed using the same antibody clone in a different buffer formulation (ab213205).

Lanes 1 - 3 : Merged signal (red and green). Green - ab213205 observed at 42 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab213205 was shown to react with CD40 in U-2 OS wild-type cells in Western blot. Loss of signal was observed when CD40 knockout sample was used. U-2 OS wild-type and CD40 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab213205 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CD40 antibody [EPR20540] (<a href='/en-us/products/primary-antibodies/cd40-antibody-epr20540-ab213205'>ab213205</a>) at 1/2000 dilution

Lane 1:

Wild-type U-2 OS whole cell lysate at 20 µg

Lane 2:

CD40 knockout U-2 OS whole cell lysate at 20 µg

Lane 2:

Western blot - Human CD40 knockout U-2 OS cell line (<a href='/en-us/products/cell-lines/human-cd40-knockout-u-2-os-cell-line-ab262486'>ab262486</a>)

Lane 3:

Raji (Human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Predicted band size: 30 kDa

Observed band size: 42 kDa

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