Pancreatic tumor sections were isolated from both PKT mice and orthotopic
xenografts and fixed in 10% neutral buffered formalin solution. Tissue slides
were then deparaffinized followed by heat induced antigen retrieval in citrate
buffer (pH = 6.0). This was followed by quenching endogenous peroxidase activity
by incubating in 3% H2O2 for ten minutes. Tissues were
further immunostained using indicated primary antibodies (Supplementary Table S1 ). Primary
antibodies were then detected using VECTASTAIN Elite ABC peroxidase kit as per
the manufacturer’s protocol using diaminobenzidine (DAB) as the
chromogen. Finally, the sections were counterstained with Mayer’s
hematoxylin and mounted with D.P.X. Tissue sections and were microscopically
examined. Digital slide images were adjusted to exclude areas containing
histologic artifacts, such as tissue folds or nonorganic material, from the
digital image. Positive staining was quantified by using ImageJ image analysis
software (NIH) and reported as percentage area of staining. Trichrome blue
staining was performed as previously described (14 (link)). PKT tumor tissues from four week endpoint analysis arm were
additionally stained using hematoxylin and eosin. Total tumor area relative to
normal pancreas was estimated in these sections by manual quantification of
random fields.
xenografts and fixed in 10% neutral buffered formalin solution. Tissue slides
were then deparaffinized followed by heat induced antigen retrieval in citrate
buffer (pH = 6.0). This was followed by quenching endogenous peroxidase activity
by incubating in 3% H2O2 for ten minutes. Tissues were
further immunostained using indicated primary antibodies (
antibodies were then detected using VECTASTAIN Elite ABC peroxidase kit as per
the manufacturer’s protocol using diaminobenzidine (DAB) as the
chromogen. Finally, the sections were counterstained with Mayer’s
hematoxylin and mounted with D.P.X. Tissue sections and were microscopically
examined. Digital slide images were adjusted to exclude areas containing
histologic artifacts, such as tissue folds or nonorganic material, from the
digital image. Positive staining was quantified by using ImageJ image analysis
software (NIH) and reported as percentage area of staining. Trichrome blue
staining was performed as previously described (14 (link)). PKT tumor tissues from four week endpoint analysis arm were
additionally stained using hematoxylin and eosin. Total tumor area relative to
normal pancreas was estimated in these sections by manual quantification of
random fields.
