Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded human liver tissue labeling IRF8 with ab306552 at 1/500 (1.082 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Negative control : no staining in human liver. The section was incubated with ab306552 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HCT116 (human colorectal carcinoma epithelial cell, Left) / THP-1 (human monocytic leukemia monocyte, Right) cells labeling IRF8 with ab306552 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. Negative control : HCT116 (PMID : 30540937).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling IRF8 with ab306552 at 1/500 (1.082 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining in human tonsil (PMID : 18663414). The section was incubated with ab306552 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized THP-1 (human monocytic leukemia monocyte) cells labeling IRF8 with ab306552 at 1/500 (1.082 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 nuclear and weak cytoplasmic staining in THP-1 cell line. Negative control : HCT116 (PMID : 30540937). 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded human Hodgkin's Lymphoma tissue labeling IRF8 with ab306552 at 1/500 (1.082 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining in human Hodgkin's Lymphoma. The section was incubated with ab306552 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded human colon tissue labeling IRF8 with ab306552 at 1/500 (1.082 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining in immune cells of human colon. The section was incubated with ab306552 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A20 (mouse reticulum sarcoma B lymphocyte) cells labeling IRF8 with ab306552 at 1/500 (1.082 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 nuclear and weak cytoplasmic 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling IRF8 with ab306552 at 1/500 (1.082 ug/ml) followed by ready to use LeicaDS9800 (Bond® Polymer Refine Detection). Positive staining in mouse spleen. The section was incubated with ab306552 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 with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A20 (mouse reticulum sarcoma B lymphocyte) cells labeling IRF8 with ab306552 at 1/50 dilution (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.

• WB

Supplier Data

Western blot - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. Negative control : human liver (PMID : 19074829; PMID : 2111015). Exposure time : Lanes 1-2 : 81 seconds, Lane 3 : 26 seconds.

All lanes:

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

Lane 1:

Human lymph node tissue lysate 20 μg

Lane 2:

Human liver tissue lysate 20 μg

Lane 3:

Mouse lymph node tissue lysate 20 μg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 48 kDa

false

Exposure time: 81s

• WB

Supplier Data

Western blot - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. Negative control : HCT 116 (PMID : 30540937; PMID : 19074829). Exposure time : Lanes 1-5 : 15 seconds, Lanes 6-7 : 81 seconds.

All lanes:

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

Lane 1:

A20 (mouse reticum sarcoma B lymphocyte) whole cell lysate 20 μg

Lane 2:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate 20 μg

Lane 3:

THP-1 (human monocytic leukemia monocyte) whole cell lysate 20 μg

Lane 4:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate 20 μg

Lane 5:

Ramos (human Burkitts lymphoma B lymphocyte) whole cell lysate 20 μg

Lane 6:

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

Lane 7:

Mouse spleen tissue lysate 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

Observed band size: 48 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-IRF8 antibody [EPR26382-10] - BSA and Azide free (AB306553)

This data was developed using ab306552, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/irf8-antibody-epr26381-181-ab307292'>ab307292</a>) 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