Anti-CD74 antibody [EPR25399-94]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD74 antibody [EPR25399-94] (AB289885)

Immunofluorescent analysis of 80% methanol fixed, 0.1% Triton X-100 permeabilized A20 (mouse reticulum sarcoma cell line) cells labeling CD74 with ab289885 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150081) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in A20 cell line. The nuclear counter stain is DAPI (blue).

Tubulin was labeled using ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).

Secondary antibody only control : PBS instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD74 antibody [EPR25399-94] (AB289885)

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling CD74 with ab289885 at 1/2000 dilution, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on mouse lung. Counter stained with Hematoxylin. The section was incubated with ab289885 for 30 mins at room temperature.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) was used.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD74 antibody [EPR25399-94] (AB289885)

Immunohistochemical analysis of paraffin-embedded mouse lung cancer tissue labeling CD74 with ab289885 at 1/2000 dilution, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on mouse lung cancer. Counter stained with Hematoxylin. The section was incubated with ab289885 for 30 mins at room temperature.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) was used.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD74 antibody [EPR25399-94] (AB289885)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling CD74 with ab289885 at 1/2000 dilution, followed by ready to use LeicaDS9800 (BOND™ Polymer Refine Detection). Positive staining on mouse spleen (PMID : 30191677). Counter stained with Hematoxylin. The section was incubated with ab289885 for 30 mins at room temperature.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins was used.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ready to use LeicaDS9800 (BOND™ Polymer Refine. Detection) was used.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-CD74 antibody [EPR25399-94] (AB289885)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse spleen (fresh) tissue labelling CD74 with ab289885 at 1/500 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution (Green). Positive staining on mouse spleen. The nuclear counterstain is DAPI (Blue).

Secondary antibody only control : Used PBS instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-CD74 antibody [EPR25399-94] (AB289885)

CD74 was immunoprecipitated from mouse spleen tissue lysate with ab289885 at 1/30 dilution (2 μg in 0.35 mg lysates). Western blot was performed from the immunoprecipitate using ab289885 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) at 1/5000 dilution was used as secondary antibody.

Lane 1 : Mouse spleen tissue lysate 10 μg (Input).

Lane 2 : ab289885 IP in Mouse spleen tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab289885 in mouse spleen tissue lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 6 seconds.

All lanes:

Immunoprecipitation - Anti-CD74 antibody [EPR25399-94] (ab289885)

Predicted band size: 34 kDa

Observed band size: 27 kDa,36 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-CD74 antibody [EPR25399-94] (AB289885)

5% NFDM/TBST was used as blocking and diluting buffer.

Exposure time :

Lane 1 : 6 seconds;

Lanes 2 : 59 seconds

All lanes:

Western blot - Anti-CD74 antibody [EPR25399-94] (ab289885) at 1/1000 dilution

Lane 1:

A20 (mouse reticulum sarcoma b lymphocyte) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 34 kDa

Observed band size: 27 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-CD74 antibody [EPR25399-94] (AB289885)

5% NFDM/TBST was used as blocking and diluting buffer.

Exposure times :

Lanes 1-2 : 10 seconds;

Lanes 3-6 : 114 seconds

The observed MW are consistent with what has been described in the literature (PMID : 19413900).

Negative control : mouse kidney (PMID : 28219888).

We observe an unknown band at around 60kDa.

All lanes:

Western blot - Anti-CD74 antibody [EPR25399-94] (ab289885) at 1/1000 dilution

Lane 1:

Mouse spleen tissue lysate at 20 µg

Lane 2:

Mouse thymus tissue lysate at 20 µg

Lane 3:

Mouse PBMC (Peripheral blood mononuclear cell) tissue lysate at 20 µg

Lane 4:

Mouse liver tissue lysate at 20 µg

Lane 5:

Mouse lung tissue lysate at 20 µg

Lane 6:

Mouse kidney tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 34 kDa

Observed band size: 27 kDa,36 kDa

false