Anti-ROR gamma antibody [Q31-378]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROR gamma antibody [Q31-378] (AB289552)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling ROR gamma with ab289552 at 1/10000 dilution, followed by the use of LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse spleen. The section was incubated with ab289552 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ROR gamma antibody [Q31-378] (AB289552)

Immunohistochemical analysis of paraffin-embedded mouse thymus tissue labeling ROR gamma with ab289552 at 1/10000 dilution, followed by the use of LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse thymus. The section was incubated with ab289552 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is LeicaDS9800 (Bond™ Polymer Refine Detection).

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ROR gamma antibody [Q31-378] (AB289552)

Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween-20-permeabilized mouse thymocytes labeling ROR gamma with ab289552 at 1/1000 dilution (Right) compared with Mouse monoclonal IgG isotype control (Left). Goat anti-Mouse IgG (Alexa Fluor^® 488, ab150113) at 1/2000 dilution was used as secondary antibody.

Cells were surface stained with APC/Fire™ 750 conjugated CD4. Then fixed with 2% PFA for 10min followed by intracellularly stained with isotype control.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ROR gamma antibody [Q31-378] (AB289552)

Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween-20-permeabilized mouse thymocytes labeling ROR gamma with ab289552 at 1/1000 dilution (Right) compared with Mouse monoclonal IgG isotype control (Left). Goat anti-Mouse IgG (Alexa Fluor^® 488, ab150113) at 1/2000 dilution was used as secondary antibody.

Cells were surface stained with Alexa Fluor^® 647 conjugated CD8. Then fixed with 2% PFA for 10min followed by intracellularly stained with isotype control.

• IP

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Immunoprecipitation - Anti-ROR gamma antibody [Q31-378] (AB289552)

ROR gamma was immunoprecipitated from mouse thymus lysate with ab289552 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab289552 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1 : Mouse thymus lysate 10 μg (Input).

Lane 2 : Mouse thymus lysate

Lane 3 : Mouse IgG2a (ab18413) monoclonal instead of ab289552 in mouse thymus lysate

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 23 seconds.

All lanes:

Immunoprecipitation - Anti-ROR gamma antibody [Q31-378] (ab289552)

Predicted band size: 58 kDa

false

Exposure time: 23s

• WB

Supplier Data

Western blot - Anti-ROR gamma antibody [Q31-378] (AB289552)

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

The expression profile is consistent with the literature : PMID 14691482 and 9881970.

All lanes:

Western blot - Anti-ROR gamma antibody [Q31-378] (ab289552) at 1/1000 dilution

Lane 1:

Mouse thymus tissue lysate at 20 µg

Lane 2:

Mouse spleen tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 58 kDa

Observed band size: 50 kDa

false

Exposure time: 10s