Recombinant Anti-ALDH1A1 antibody [EP1933Y] - C-terminal (ab52492)

Blocking/Diluting buffer 5% NFDM/TBST

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.

ab52492 (purified) at 1/20 dilution (2ug) immunoprecipitating ALDH1A1 in HepG2 whole cell lysates.
Lane 1: HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysates 10ug
Lane 2 (+): ab52492 & 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.

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).

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.

Sections of the proximal (top) and distal (bottom) mouse colon were assayed for protein expression of ALDH1A1.

Signals yielded by immunohistochemical staining (red) are shown in merged images with DAPI staining (blue). Representative data are shown for three independent experiments. Arrowheads show positive cells for ALDH1A1 in the distal colon. Scale bars, 50 μm.

Colonic tissues were fixed overnight at 4°C in 4% paraformaldehyde, sequentially dehydrated in 10, 15 and 20% sucrose in PBS, and embedded in OCT compound. Colonic organoids were fixed together with surrounding collagen type I gel and processed in the same manner. Frozen sections of 8-μm thickness were subjected to immunohistochemistry using ab52492 (unpurified). 

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

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

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

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

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.