Anti-IRF8 antibody [EPR26381-181]

• IHC

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Immunohistochemistry - Anti-IRF8 antibody [EPR26381-181] (AB307292)

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling IRF8 with ab307292 at 1/500 (1.06 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Negative control : almost no staining on rat cerebrum. The section was incubated with ab307292 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC

Supplier Data

Immunohistochemistry - Anti-IRF8 antibody [EPR26381-181] (AB307292)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling IRF8 with ab307292 at 1/500 (1.06 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Negative control : almost no staining on mouse cerebrum. The section was incubated with ab307292 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC

Supplier Data

Immunohistochemistry - Anti-IRF8 antibody [EPR26381-181] (AB307292)

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling IRF8 with ab307292 at 1/500 (1.06 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining on rat spleen. The section was incubated with ab307292 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC

Supplier Data

Immunohistochemistry - Anti-IRF8 antibody [EPR26381-181] (AB307292)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling IRF8 with ab307292 at 1/500 (1.06 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining on mouse spleen. The section was incubated with ab307292 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC

Supplier Data

Immunohistochemistry - Anti-IRF8 antibody [EPR26381-181] (AB307292)

Immunohistochemical analysis of paraffin-embedded mouse Burkitt's lymphoma tissue labeling IRF8 with ab307292 at 1/500 (1.06 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining on mouse Burkitt's lymphoma. The section was incubated with ab307292 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IRF8 antibody [EPR26381-181] (AB307292)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A20 (mouse reticulum sarcoma B lymphocyte) cells labeling IRF8 with ab307292 at 1/100 (5.3 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing mainly nuclear staining in A20 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 ug/ml) dilution (Red). The nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IRF8 antibody [EPR26381-181] (AB307292)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A20 (mouse reticulum sarcoma B lymphocyte) cells labeling IRF8 with ab307292 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-IRF8 antibody [EPR26381-181] (AB307292)

IRF8 was immunoprecipitated from 0.35 mg A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate 10 ug with ab307292 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307292 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : A20 whole cell lysate 10 ug Lane 2 : ab307292 IP in A20 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307292 in A20 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 76 seconds.

All lanes:

Immunoprecipitation - Anti-IRF8 antibody [EPR26381-181] (ab307292) at 1/1000 dilution

Lane 1:

A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate 10 μg

Lane 2:

A20 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 48 kDa

false

Exposure time: 76s

• WB

Supplier Data

Western blot - Anti-IRF8 antibody [EPR26381-181] (AB307292)

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

Negative and positive control : (PMID : 2111015).

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-IRF8 antibody [EPR26381-181] (ab307292) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse spleen 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 1/100000 dilution

Observed band size: 48 kDa

true

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-IRF8 antibody [EPR26381-181] (AB307292)

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

Negative and positive control : (PMID : 2111015).

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-IRF8 antibody [EPR26381-181] (ab307292) at 1/1000 dilution

Lane 1:

Rat brain tissue lysate at 20 µg

Lane 2:

Rat spleen 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 1/100000 dilution

Observed band size: 48 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-IRF8 antibody [EPR26381-181] (AB307292)

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

Compared to ab306552, ab307292 demonstrates lower sensitivity in Western blot.

We suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results when using ab307292 in western blot.

Lanes 1 - 3:

Western blot - Anti-IRF8 antibody [EPR26381-181] (ab307292) at 1/1000 dilution

Lanes 4 - 6:

Western blot - Anti-IRF8 antibody [EPR26382-10] (<a href='/en-us/products/primary-antibodies/irf8-antibody-epr26382-10-ab306552'>ab306552</a>) at 1/1000 dilution

Lanes 1 and 4:

Mouse spleen tissue lysate at 20 µg

Lanes 2 and 5:

Mouse lymph node tissue lysate at 20 µg

Lanes 3 and 6:

A20 (mouse reticum sarcoma B lymphocyte) 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 1/20000 dilution

Observed band size: 48 kDa

false

Exposure time: 60s