![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](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_1/25650_ab64772-195129-IPV021ab6477212min.jpg)
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
Anti-CD74 antibody (ab64772) at 1 µg/ml + Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 10 µgSecondaryGoat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilutionPerformed under reducing conditions.
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
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.See Abreview
ICC/IF image of ab64772 stained RAW246.7 cells. The cells were 4% paraformaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab64772, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.