Anti-Bcl6 antibody [GI191E]

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl6 antibody [GI191E] (AB241549)

The BOND™ Polymer Refine Detection System on the BOND RX Automated Stainer (DS9800, Leica Biosystems, Wetzlar, Germany) was used for immunohistochemical labelling of formalin-fixed, paraffin-embedded (FFPE) tissues. The protocol includes a peroxide block, post-primary reagent, polymer detection, DAB chromogen, and haematoxylin counterstain, all automated to minimize variability. Antigen retrieval was performed by heat-induced epitope retrieval (HIER) for 20 minutes in Tris-EDTA buffer (BOND Epitope Retrieval Solution 2, AR9640, Leica Biosystems, Wetzlar, Germany). For mouse and rabbit antibodies, the sequence comprised peroxide block (10 min), primary antibody incubation (30 min), post-primary (10 min), polymer (10 min), DAB (10 min), and haematoxylin (8 min). For rat antibodies, an anti-rat step (1 : 300 dilution, Vector Laboratories, California, USA; 20 min) was included prior to polymer application (15 min).

For mouse tissues, antigen retrieval was performed using high pH buffers (Dako, Agilent), followed by blocking of endogenous peroxidase activity with 3% hydrogen peroxide. Slides were then incubated with the primary antibody. Antigen/antibody complexes detection was carried out using a horseradish peroxidase-conjugated visualization system (Novolink Polymer, Leica). Staining was developed with 3,3′-diaminobenzidine (EnVision FLEX DAB Enhancer, Dako, Agilent), and nuclei were counterstained with hematoxylin (FLEX Hematoxylin, SM806, Dako, Agilent). Images were acquired using an Axiocam CCD camera (Zeiss) and processed with ZEN 2.1 software and Photoshop 9.0.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl6 antibody [GI191E] (AB241549)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling Bcl6 with ab241549 at 1/10000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Nuclear staining on the germinal center in the human tonsil. The section was incubated with ab241549 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND ^® RX instrument

The section was counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl6 antibody [GI191E] (AB241549)

Immunohistochemical analysis of paraffin-embedded Human B cell lymphoma tissue labelling Bcl6 with ab241549 at 1/10000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Nuclear staining on the human B cell lymphoma. The section was incubated with ab241549 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND ^® RX instrument

The section was counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-Bcl6 antibody [GI191E] (AB241549)

Bcl6 was immunoprecipitated from 1mg of Ramos (Human Burkitt's lymphoma cell line) whole cell lysate with ab241549 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab241549 at 1/500 dilution. Anti-mouse IgG for IP (HRP) (ab131368) was used at 1/1000 dilution.

Lane 1 : Ramos whole cell lysate 10μg (Input).
Lane 2 : ab241549 IP in Ramos whole cell lysate.
Lane 3 : Mouse monoclonal IgG instead of ab241549 in Ramos whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-Bcl6 antibody [GI191E] (ab241549)

Predicted band size: 79 kDa

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl6 antibody [GI191E] (AB241549)

Immunohistochemical analysis of paraffin-embedded Mouse mesenteric lymph nodes tissue labelling Bcl6 with ab241549 at 1/10000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Nuclear staining on the germinal center in the mouse mesenteric lymph nodes. The section was incubated with ab241549 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND ^® RX instrument

The section was counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bcl6 antibody [GI191E] (AB241549)

Immunohistochemical analysis of paraffin-embedded Rat mesenteric lymph nodes tissue labelling Bcl6 with ab241549 at 1/10000 dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Nuclear staining on the germinal center in the rat mesenteric lymph nodes. The section was incubated with ab241549 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND ^® RX instrument

The section was counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• WB

Supplier Data

Western blot - Anti-Bcl6 antibody [GI191E] (AB241549)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Bcl6 antibody [GI191E] (ab241549) at 1/1000 dilution

Lane 1:

Ramos (Human Burkitt’s lymphoma B lymphocyte), whole cell lysate at 20 µg

Lane 2:

Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 79 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Bcl6 antibody [GI191E] (AB241549)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure times : Lane 1,3 : 81 secs; Lane 2 : 5 secs.

All lanes:

Western blot - Anti-Bcl6 antibody [GI191E] (ab241549) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse heart tissue lysate at 20 µg

Lane 3:

Mouse kidney tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 79 kDa

false