Anti phospho fak tyr 397

Colon samples for immunofluorescence were swiss-rolled and fixed in 10% formalin and then processed and embedded in paraffin. 5-micron sections were cut and underwent a deparaffination process followed by a citrate antigen retrieval step before being permeabilized in 0.5% triton-X 100 in PBS and blocked with 5% normal goat serum. Primary antibodies include, from Cell Signaling Technology (Danvers, MA), anti-Phospho-p44/42 MAPK cat#4370, anti-phospho-FAK (Tyr397) cat#3283. Samples were incubated in primary antibody overnight at 4ºC. Samples were then washed three times and incubated for 1 hour in fluorochrome-conjugated secondary antibody. Samples were again washed and incubated with DAPI at a concentration of 1:10000 in PBS for 5 minutes. Samples were mounted using Prolong diamond antifade and imaged on an Olympus FV1000 confocal microscope at 20X. At least 3 sections and three different images were taken for each sample. For immunoblot analysis, immunoreactive species were detected using the aforementioned anti-Phospho-p44/42 MAPK cat#4370, anti-phospho-FAK (Tyr397) cat#3283, followed by anti-rabbit HRP or anti-mouse HRP followed by visualization with ECL chemiluminescence detection reagent (GE Healthcare Biosciences Piscataway NJ). Experiments were conducted in triplicate and the average density of the band quantified using ImageJ software.