重组Anti-CD74抗体[EPR4064] (ab108393)

Lanes 1 - 4: Merged signal (red and green). Green - ab108393 observed at 35 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab108393 was shown to react with CD74 in western blot. The band observed in CD74 CRISPR/Cas9 edited cell line ab273378 (CRISPR/Cas9 edited lysate ab275529) below 35 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab108393 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 h at room temperature before imaging.

IHC image of CD74 staining in a section of frozen human normal tonsil* performed on a Leica Biosystems BOND® RX instrument using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab108393, 0.05ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Negative control image: IHC image of CD74 staining in a section of frozen human normal heart* performed on a Leica Biosystems BOND® RX instrument using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab108393, 0.05ugml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Lanes 1 - 4: Merged signal (red and green). Green - ab108393 observed at 34 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab108393 was shown to react with CD74 in wild-type Raji cells in Western blot with loss of signal observed in CD74 knockout cell line ab273876 (knockout cell lysate ab273830). Wild-type Raji and CD74 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab108393 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 h at room temperature before imaging.

ELISA using ab108393 at varying antibody concentrations and antigen concentration at 1000 ng/mL. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) was used as the secondary antibody.

Immunohistochemical analysis of CD74 in paraffin embedded Human tonsil tissue, using ab108393 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Intracellular Intracellular Flow Cytometry overlay histogram showing wild-type Raji (green line) and CD74 knockout Raji cells (ab273378) stained with ab108393 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10µg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab108393) (1x10⁶ in 100µl at 0.2 µg/ml) for 30 min at 22°C. 

The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluorr^® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 22°C. Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type Raji cells - black line; CD74 knockout Raji cells ab273378 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). 

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. 

Confocal image of ab108393 staining CD74 in the RAMOS (human Burkitt's lymphoma) cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Samples were incubated with primary antibody (1/500) and an Alexa Fluor® 488-conjugated goat anti-rabbit  IgG polyclonal (ab150077) was used as the secondary antibody at a dilution of 1/1000.

DAPI was used ad a nuclear counterstain at 1/200.

ab108393staining CD74 inRAMOS (human Burkitt's lymphoma) cell line.The sample was fixed with 4% paraformaldehyde and incubated with the primary antibody (1/150) for 30 minutes at 4°C. AnAlexa Fluorr^® 488 -conjugated goatanti-rabbit IgG (1/2000) was used as the secondary antibody. 

Control:Cell without incubation with primary antibody and secondary antibody (Blue). 

ab108393 showing negative staining in Normal brain tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab108393 showing positive staining in B cell lymphoma tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab108393 showing negative staining in Normal placenta tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab108393 showing positive staining in Normal spleen tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab108393 showing negative staining in Normal heart tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab108393 showing negative staining in Normal liver tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.