All lanes : Anti-Visfatin antibody [EPR21980] (ab236874) at 1/1000 dilution

Lane 1 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 20 µg
Lane 2 : Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 20 µg
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 4 : HT-1080 (human fibrosarcoma epithelial cell), whole cell lysate at 20 µg
Lane 5 : PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 6 : Human fetal kidney lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 55 kDa

Blocking/Diluting buffer and concentration: 5% NFDM/TBST 

Exposure times:
Lanes 1-2: 26 seconds
Lanes 3-5: 92 seconds
Lane 6: 3 minutes

Lane 6 was developed using a higher sensitivity ECL substrate.

The molecular weight observed is consistent with what has been described in the literature (PMID:25368373, PMID:12782293).

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling Visfatin using ab236874 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear and cytoplasmic staining in human bladder cancer (PMID:26396920) is observed. Counterstained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (human colorectal carcinoma epithelial cell) cells labeling Visfatin (Green) with ab236874 at 1/100 dilution, followed by AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. Confocal image showing cytoplasmic and nuclear staining in HCT 116 cell line is observed. The nuclear counterstain is DAPI (Blue). Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as the counterstain (Red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Alexa-Fluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

Visfatin was immunoprecipitated from 0.35 mg of K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab236874 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab236874 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

Lane 1: K562 whole cell lysate 10 μg (input).
Lane 2: ab236874 IP in K562 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab236874 in K562 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HCT 116 (human colorectal carcinoma epithelial cell) cell line labeling Visfatin with ab236874 at 1/500 dilution (Red) compared with the Rabbit monoclonal IgG (ab172730, Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody, Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized K562 (human chronic myelogenous leukemia lymphoblast) cell line labeling Visfatin with ab236874 at 1/500 dilution (Red) compared with the Rabbit monoclonal IgG (ab172730, Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody, Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Visfatin using ab236874 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear and cytoplasmic staining in rat liver (PMID: 29104634; PMID:24603648) is observed. Counterstained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Visfatin using ab236874 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear and cytoplasmic staining in mouse kidney (PMID: 29104634; PMID:24287278) is observed. Counterstained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling Visfatin using ab236874 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Nuclear and cytoplasmic staining in human breast cancer (PMID:21784959) is observed. Counterstained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-Visfatin antibody [EPR21980] (ab236874) at 1/1000 dilution

Lane 1 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 2 : HCT 116 (human colorectal carcinoma epithelial cell), whole cell lysate at 20 µg
Lane 3 : HT-1080 (human fibrosarcoma epithelial cell), whole cell lysate at 20 µg
Lane 4 : C6 (rat glial tumor glial cell), whole cell lysate at 20 µg
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 20 µg
Lane 6 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 55 kDa

Blocking/Diluting buffer and concentration: 5% NFDM/TBST

Exposure time:
Lanes 1-3: 40 seconds
Lanes 4-5: 3 minutes
Lane 6: 15 seconds

The molecular weight observed is consistent with what has been described in the literature (PMID:25368373, PMID:12782293).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (human chronic myelogenous leukemia lymphoblast) cells labeling Visfatin (Green) with ab236874 at 1/100 dilution, followed by AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution. Confocal image showing cytoplasmic and nuclear staining in K562 cell line is observed. The nuclear counterstain is DAPI (Blue). Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as the counterstain (Red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Alexa-Fluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.