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

All lanes : Anti-CD40 antibody [EPR20540] (ab213205) at 1/2000 dilution

Lane 1 : Wild-type U-2 OS whole cell lysate
Lane 2 : CD40 knockout U-2 OS whole cell lysate
Lane 3 : Raji whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 30 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?

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.

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.

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.