Aldh1 antibody

Tissues from 68 patients were included in the tissue microarray. These patients had vertical growth phase primary melanomas and no regional nodal procedure or apparent metastases at the time of definitive treatment between 1972 and 1991 at the University of Pennsylvania’s Pigmented Lesion Clinic. The tissue microarray was composed of 2 mm cores. When we designed the tissue microarray, we considered the possibility of heterogeneity within melanomas. Sixty three percent of cases had three cores from different areas of primary melanomas and thirty four percent of the cases had four cores. Two cases had one or two cores due to smaller primary tumor volumes. Immunohistochemical staining was performed for ALDH1 antibody (BD biosciences) using a 1:800 dilution with citrate retrieval. ALDH1 expression was analyzed by two independent pathologists (RMA, XX) using a modified histological score (H-score) based both on the percentage of positively stained cells and on the intensity of staining, with a maximum score of 300. For statistical analysis, the scores for each core reported by the two readers were averaged. The tissue microarray was constructed at the Penn tissue microarray facility in the Department of Pathology and Laboratory Medicine.

Briefly, placentas were weighed, divided into a maternal or fetal aspect, frozen, then homogenized using a mortar and pestle. Tissue was resuspended in RIPA buffer containing protease and phosphatase inhibitors (Millipore Sigma) at 1mg/ml. 5 micrograms of each lysate was loaded per lane. Protein concentration was determined by using the Bio-Rad dye-binding assay. Western blotting was performed as described previously (n=5–7/group) (23 ). Briefly, the solubilized protein homogenates were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Transblot; Bio-Rad, Hercules, CA). The following primary antibodies were used for signal detection: ALDH1 antibody (cat# 611195, BD Biosciences, City, San Jose, CA, USA; ~55 kDA; 1:1000 dilution) and ALDH2 antibody (cat #ab108306, Abcam, Cambridge, MA; ~56 kDA; 1:1000 dilution). Anti-beta-actin antibody (cat #A0760–40, US Biological, Salem, MA; ~42kDA; 1:20,000 dilution) was used to detect endogenous beta actin expression, which served as an internal control for inter-lane loading variability. The quantification of protein bands was performed by densitometry using ImageJ software, as previously described (23 ).