Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human pancreas tissue sections labeling Islet 1 with purified ab178400 at 1/6000 dilution (0.03 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-Islet 1 antibody [EPR10362] (ab178400) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Islet 1 knockout HAP1 whole cell lysate
Lane 3 : SH-SY5Y whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 39 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab178400 observed at 40 kDa. Red - loading control, ab18058, observed at 130 kDa.

Unpurified ab178400 was shown to recognize Islet 1 in wild-type HAP1 cells as signal was lost at the expected MW in Islet 1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Islet 1 knockout samples were subjected to SDS-PAGE. Ab178400 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1,000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Islet 1 antibody [EPR10362] (ab178400) at 1/10000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 39 kDa
Observed band size: 39 kDa

ab178400 (purified ) at 1/20 dilution (1ug) immunoprecipitating Islet 1 in SH-SY5Y whole cell lysate.
Lane 1 (input): SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate 10ug
Lane 2 (+): ab178400 & SH-SY5Y whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab178400 in SH-SY5Y whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling Islet 1 with ab178400  followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on human pancreatic islet. The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Anti-Islet 1 antibody [EPR10362] (ab178400) at 1/10000 dilution ((unpurified)) + A375 (HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 39 kDa

Immunohistochemical analysis of formalin-fixed, paraffin-embedded, human gastric neuroendocrine carcinoma tissue labeling Islet 1 with unpurified ab178400 at a 1/250 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Islet 1 was immunoprecipitated from 0.35 mg SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate 10μg with unpurifed ab178400 at 1:50 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using unpurified ab178400 1:1000 dilution (2 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1:1000 dilution.

Lane 1: SH-SY5Y (Human neuroblastoma epithelial cell) whole cell lysate 10μg

Lane 2: ab178400 IP in SH-SY5Y whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab178400 in SH-SY5Y whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST. Exposure time: 1 second

All lanes : Anti-Islet 1 antibody [EPR10362] (ab178400) at 1/1000 dilution ((unpurified))

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 2 : K562 (human chronic myelogenous leukemia cell line from bone marrow ) cell lysate
Lane 3 : Jurkat (human T cell leukemia cell line from peripheral blood) cell lysate
Lane 4 : SH-SY5Y (human neuroblastoma cell line from bone marrow) cell lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 39 kDa

Immunohistochemical analysis of formalin-fixed, paraffin-embedded, human small cell lung carcinoma tissue labeling Islet 1 with unpurified ab178400 at a 1/250 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Western blot analysis on Immunoprecipitation pellet from either Lane 1) SH-SY5Y cell lysate, or Lane 2) 1xPBS (negative control); showing Islet 1, using unpurifed ab178400 at 1/10 dilution and HRP-conjugated anti-rabbit IgG preferentially detecting the non-reduced form of rabbit IgG.