Publication protocol
"Tissue samples were drop-fixed in 10% buffered formalin for 48 hours prior to paraffin embedding. Samples were cut into 4 µm sections, deparaffinized, and washed with
PBS. For antigen retrieval, samples were microwaved for 12 minutes in pH6 sodium citrate buffer (Cyclin D1, PanCK, GS, CD45) or Tris-EDTA buffer (Ki67), or were
pressure cooked for 20 minutes in pH6 sodium citrate buffer (β-catenin), Dako Target Retrieval Solution (Dako, S1699) (CK19, EpCAM), or pH9 EDTA buffer (αSMA). After
cooling, samples were placed in 3% H2O2 for 10 minutes to quench endogenous peroxide activity. After washing with PBS, slides were blocked with Super Block
(ScyTek Laboratories, AAA500) for 10 minutes or 10% goat serum in PBS for 10 minutes (GS, p21). The primary antibodies were incubated at the following
concentrations in antibody diluent (PBS + 1% BSA (Fisher BioReagents, BP1605-100) with 0.1% Tween™ 20 (Fisher BioReagents, BP337-500)): GS (Sigma G2781, 1:1500),
Ki67 (Thermo Scientific RM-9106-S, 1:100), PanCK (Dako Z0622, 1:200), Cyclin D1 (Abcam ab134175, 1:200), β-catenin (Abcam ab32572, 1:100) for one hour at room
temperature or at 4℃ overnight: p21 (Santa Cruz sc-471, 1:25), EpCAM (Biolegend 118201, 1:50), CK19 (DSHB TROMA III, 1:10). Samples were washed with PBS three
times and incubated with the appropriate biotinylated secondary antibody (Vector Laboratories) diluted 1:500 or 1:1000 (GS) in antibody diluent for 30 minutes at room
temperature. Samples were washed with PBS three times and sensitized with the Vectastain® ABC kit (Vector Laboratories, PK-6101) for 30 minutes. Following three
washes with PBS color was developed with DAB Peroxidase Substrate Kit (Vector Laboratories, SK-4100), followed by quenching in distilled water for five minutes. Slides
were counterstained with hematoxylin (Thermo Scientific, 7211), dehydrated to xylene and coverslips applied with Cytoseal™ XYL (Thermo Scientific, 8312-4). For H&E
staining, samples were deparaffinized and stained with hematoxylin (Thermo Scientific, 7211) and eosin (Thermo Scientific, 71204), followed by dehydration to xylene and
application of a coverslip. For Sirius Red staining, samples were deparaffinized and incubated for one hour in Picro-Sirius Red Stain (American MasterTech, STPSRPT),
washed twice in 0.5% acetic acid water, dehydrated to xylene, and coversliped. Images were taken on a Zeiss Axioskop 40 inverted brightfield microscope. Images for tiling
were taken on a Zeiss Axio Observer.Z1 microscope and assembled utilizing ZEN Imaging software. "
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