Anti-CD74 antibody [PIN.1]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD74 antibody [PIN.1] (AB22603)

ab22603 staining CD74 in human liver. Staining is localized to the membrane and cytoplasm.
Left panel : with primary antibody at 4ug/ml. Right panel : isotype control.
Sections were stained using an automated system (Dako Autostainer plus), at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer, EDTA pH 9.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be req

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD74 antibody [PIN.1] (AB22603)

ICC/IF image of ab22603 stained HepG2 cells. The cells were 100% methanol fixed (5 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 (ab22603, 10μg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse 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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD74 antibody [PIN.1] (AB22603)

ICC/IF image of ab22603 staining CD74 in HaCaT cells. Cells were fixed with cold 100% methanol for 10 minutes at -20°C. Samples were incubated with primary antibody at 1 : 100 dilution for 1 hour at room temperature. A FITC Goat anti-mouse (green) was used as a secondary antibody at 1 : 50 dilution for 1 hour at room temperature.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD74 antibody [PIN.1] (AB22603)

Immunofluorescent analysis of 2% Formaldehyde-fixed Cervical cancer cell line (HeLa) labeling CD74 with ab22603 at 1/100 dilution. Secondary antibody : FITC Goat Anti-Mouse (green) at 1/200 dilution. (A) DAPI (blue) nuclear stain. (B) Anti-CD74 Antibody. (C) Composite. (A) DAPI (blue) nuclear stain. (B) Anti-CD74 Antibody. (C) Composite.

• WB

Lab

Western blot - Anti-CD74 antibody [PIN.1] (AB22603)

Lanes 1 - 4 : Merged signal (red and green). Green - ab22603 observed at 35 kDa. Red - loading control, ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55 kDa.

ab22603 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 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab22603 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4 °C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-CD74 antibody [PIN.1] (ab22603) at 1 µg/mL

Lane 1:

Wild-type Raji cell lysate at 30 µg

Lane 2:

Western blot - Human CD74 knockout Raji cell lysate (<a href='/en-us/products/cell-lysates/human-cd74-knockout-raji-cell-lysate-ab275529'>ab275529</a>)

Lane 2:

CD74 knockout Raji cell lysate at 30 µg

Lane 2:

Western blot - Human CD74 knockout Raji cell line (<a href='/en-us/products/cell-lines/human-cd74-knockout-raji-cell-line-ab273378'>ab273378</a>)

Lane 3:

Jurkat cell lysate at 30 µg

Lane 4:

HepG2 cell lysate at 30 µg

Predicted band size: 34 kDa

Observed band size: 35 kDa

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