ab68156, at 1/100 dilution, staining STAT4 in paraffin embedded human tonsil tissue sections by Immunohistochemistry

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-STAT4 antibody [EP1900Y] (ab68156) at 1/1000 dilution

Lane 1 : Daudi cell lysate
Lane 2 : K562 cell lysate
Lane 3 : Hela cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 86 kDa
Observed band size: 86 kDa
Additional bands at: 30 kDa. We are unsure as to the identity of these extra bands.

Immunocytochemistry analysis of HeLa (human cervix adenocarcinoma epithelial cell) labeling STAT4 with purified ab68156 at 1/50 dilution (9 µg/ml). Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/1000 (2 µg/ml) was used as the secondary antibody. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.16 µg/ml) was used as counterstain. Nuclei were stained blue with DAPI.
Negative control: PBS instead of the primary antibody.

Purified ab68156 at 1/30 dilution (2µg) immunoprecipitating STAT4 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab68156 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab68156 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 86 kDa

Flow Cytometry analysis of K562 (Human chronic myelogenous leukemia lymphoblast) cells labeling STAT4 with purified ab68156 at 1/100 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cells without incubation with primary antibody and secondary antibody (Blue).