Perk5 antibody

Human mesothelioma tissue arrays (obtained from Dr. Harvey Pass, NYU) were used for pERK5 staining. The array contained 15 mesothelioma sections from different patients with pleural mesothelioma. A section of normal lung was also part of the array stained for pERK5. Arrays were deparaffinized and after antigen retrieval, were blocked, washed and incubated overnight at 4°C with diluted pERK5 antibody (Cell Signaling Technology, Danvers, MA) as previously described [16 (link)]. For a negative control, one slide with all sections was stained as described, excluding primary antibody. After further washing, tissue specimens were incubated with a fluorescently conjugated secondary antibody, AlexaFluor^® 647 (Thermo Fisher, Grand Island, NY). Following nuclei staining with DAPI (Thermo Fisher), sections were mounted, coverslipped and imaged with a Zeiss 510 META laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). For quantitation fluorescent images of pERK5 (red channel only) were analyzed in MetaMorph imaging software to determine the area of positive staining and average intensity in the field of view. From this data, the average intensity within the positively stained area of pERK5 was determined for each group.