Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling ALDH1A1 with purified ab52492 at 1/50 dilution (3.54 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-ALDH1A1 antibody [EP1933Y] - C-terminal (ab52492) at 1/1000 dilution (Purified)

Lane 1 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates at 20 µg
Lane 2 : Human small intestine lysates
Lane 3 : PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysates at 20 µg
Lane 4 : MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg
Lane 5 : Mouse liver lysates at 20 µg
Lane 6 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

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

Predicted band size: 55 kDa
Observed band size: 55 kDa

Immunohistochemical analysis of Formalin/PFA-fixed paraffin-embedded normal human kidney tissue sections labeling ALDH1A1 with ab52492 (unpurified).

All lanes : Anti-ALDH1A1 antibody [EP1933Y] - C-terminal (ab52492) at 1/1000 dilution (unpurified)

Lane 1 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 2 : Human small intestine tissue lysate
Lane 3 : PC-3 (Human prostate adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 5 : Mouse liver tissue lysate
Lane 6 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 55 kDa
Observed band size: 55 kDa


Exposure time: 1 minute

Blocking/Diluting buffer 5% NFDM/TBST

Immunohistochemical analysis of Formalin/PFA-fixed paraffin-embedded normal human brain tissue sections labeling ALDH1A1 with ab52492 (unpurified).

Flow Cytometry analysis of HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling ALDH1A1 with purified ab52492 at 1/20 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 - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunohistochemical analysis of Formalin/PFA-fixed paraffin-embedded normal human lung tissue sections labeling ALDH1A1 with ab52492 (unpurified).

Tumor tissues of primary invasive ductal carcinomas of the breast were obtained from 192 female patients with stage IIB and III prior to pre-operative neoadjuvant chemotherapy.

(progressive or stable disease, PD/SD)

(partial or complete remission, PR/CR)

The level of ALDH1 was tested by immunohistochemistry staining in paraffin-embedded tissue sections. Rabbit monoclonal ALDH1A1 antibody (unpurified ab52492, Abcam) used at a 1:100 dilution.

Immunohistochemical analysis of paraffin-embedded human liver tissue sections labeling ALDH1A1 with ab52492 (unpurified) at 1/100 dilution.

ab52492 (purified) at 1/20 dilution (2µg) immunoprecipitating ALDH1A1 in HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates.
Lane 1: HepG2 whole cell lysates 10µg.
Lane 2: ab52492 IP in HepG2 whole cell lysates.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab52492 in HepG2 whole cell lysates.
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Overlay histogram showing HepG2 (Human liver hepatocellular carcinoma cell line) cells stained with ab52492 (unpurified) (red line).

The cells were fixed with 80% methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52492, 1/1000 dilution) for 30 minutes at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 minutes at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Immunocytochemistry/ Immunofluorescence analysis of HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling ALDH1A1 with ab52492 (purified) at 1/500 dilution (4 μg/ml).

Cells were fixed in 100% methanol. ab150077, an AlexaFluor^®488 Goat anti-Rabbit secondary antibody was used at 1/1000 dilution (2 μg/ml). ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain at 1/200 dilution (2.5 μg/ml). DAPI was used as nuclear counterstain.

Confocal image showing cytoplasmic staining on HepG2 cell line.

Negative control: No staining on MCF-7 cell line.