Lanes 1 - 4: Merged signal (red and green). Green - ab64772 observed at 35 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab64772 was shown to react with CD74 in western blot. The band observed in CD74 knockout cell line ab273378 (knockout 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 ab64772 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at 1 µg/ml 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.

ab64772 (1:160) staining CD74 in paraffin-embedded human tonsil (left panel) using an automated system (Ventana Discovery). Right-hand panel shows negative control (no primary antibody).
Using this protocol there is strong membrane staining of activated B cells in the germinal centres and B cells of the mantle zone of the follicles plus scattered cells of the interfollicular areas (paracortical B cells).

Sections were rehydrated and antigen retrieved in CC1 Cell Conditioning Buffer using Ventana Mild Retrieval programme. Slides were blocked in 3% H₂O₂ / 4 min / 37°C and incubated with ab64772 (1:160 dilution / 2 hours / 37°C). Sections then blocked (4mins / 37°C) and incubated with Dako swine anti-rabbit antibody (1:50, 28 min / 37°C). Staining was amplified and detected by incubation with Ventana Streptavidin ABC system (16 min / 37°C) and Ventana DAB map reagent (8 min / 37°C). Slides were counterstained with Haematox

CD74 was immunoprecipitated using 0.5mg Raji whole cell extract, 5µg of Rabbit polyclonal to CD74 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, Raji whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab64772.

Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band: 34kDa; CD74

ab64772 (1:80) staining CD74 in paraffin-embedded human lymph node (left panel) using an automated system (Ventana Discovery). Right-hand panel shows negative control (no primary antibody).
Using this protocol there is strong membrane staining of activated B cells in the germinal centres and B cells of the mantle zone of the follicles plus scattered cells of the interfollicular areas (paracortical B cells).

Sections were rehydrated and antigen retrieved in CC1 Cell Conditioning Buffer using Ventana Mild Retrieval programme. Slides were blocked in 3% H₂O₂ / 4 min / 37°C and incubated with ab64772 (1:80 dilution / 1 hour / 37°C). Sections then blocked (3mins / 37°C) and incubated with Dako swine anti-rabbit antibody (1:50, 28 min / 37°C). Staining was amplified and detected by incubation with Ventana Streptavidin ABC system (16 min / 37°C) and Ventana DAB map reagent (8 min / 37°C). Slides were counterstained with Haematoxyli

ab64772 (1/250) staining CD74 in paraffin-embedded Human tonsil tissue.  Tissue underwent fixation in formaldehyde, peroxidase blocking, protein blocking and heat mediated antigen retrieval.  The secondary antibody was goat anti rabbit conjugated to HRP.  For further experimental details please refer to abreview.

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