Spectral characteristics of AEC, DAB and NovaRed were displayed with the different color models using color bars that were generated from a range of representative IHC staining derived from the xenograft studies described below, as well as additional IHC archived slides that showed a greater range of staining intensity.
Changes in STAT3 phosphorylation (p-STAT3) levels were associated with different conditions, including growth factor stimulation and sampling techniques as previously described [16 ]. Formalin-fixed paraffin embedded specimens were obtained to compare IHC staining using different chromogens AEC, DAB or NovaRed, and hematoxylin counterstain. Briefly, a tumor xenograft was excised and divided into two equal parts. One part was further divided and subjected to immediate formalin fixation, or protein extraction for Western blot analysis [17 (link)]. The second part was incubated with 50 ng/ml epidermal growth factor (EGF, Sigma-Aldrich, Oakville, ON) in PBS for 20 minutes prior to specimen analysis. The antibody S727p-STAT3 (BD Biosciences, Mississauga, ON) was used for Western blotting at a dilution of 1:100, and anti-GAPDH used at a dilution of 1:5000 (Ambion, Foster City, CA). Protein bands were detected with ECL plus fluorescence, imaged with a Typhoon system and their intensities were quantified (mean intensity × band area) and normalized with GAPDH using ImageQuant5.2 software (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
Second, p-STAT3 levels were also quantified in formalin-fixed paraffin embedded sections of fine-needle samples obtained from a tumor xenograft. These samples were subjected to either immediate or delayed fixation after 5, 20 or 60 minutes, which caused an increase in p-STAT3.
Changes in STAT3 phosphorylation (p-STAT3) levels were associated with different conditions, including growth factor stimulation and sampling techniques as previously described [16 ]. Formalin-fixed paraffin embedded specimens were obtained to compare IHC staining using different chromogens AEC, DAB or NovaRed, and hematoxylin counterstain. Briefly, a tumor xenograft was excised and divided into two equal parts. One part was further divided and subjected to immediate formalin fixation, or protein extraction for Western blot analysis [17 (link)]. The second part was incubated with 50 ng/ml epidermal growth factor (EGF, Sigma-Aldrich, Oakville, ON) in PBS for 20 minutes prior to specimen analysis. The antibody S727p-STAT3 (BD Biosciences, Mississauga, ON) was used for Western blotting at a dilution of 1:100, and anti-GAPDH used at a dilution of 1:5000 (Ambion, Foster City, CA). Protein bands were detected with ECL plus fluorescence, imaged with a Typhoon system and their intensities were quantified (mean intensity × band area) and normalized with GAPDH using ImageQuant5.2 software (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
Second, p-STAT3 levels were also quantified in formalin-fixed paraffin embedded sections of fine-needle samples obtained from a tumor xenograft. These samples were subjected to either immediate or delayed fixation after 5, 20 or 60 minutes, which caused an increase in p-STAT3.
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