Diaminobenzidene

Tumors were fixed in 4% PFA immediately after excision from the CAM. Tumor processing (paraffin embedding, sectioning and hematoxylin/eosin staining) was performed by the Université de Sherbrooke histology core platform. Tissue sections were rehydrated and treated with 0.01 mol/L citrate, pH 6.0, and immunohistochemical staining was performed according to the standard avidin-biotin immunoperoxidase complex technique using anti-KI67 (1:200, GTX16667, Genetex, Irvine, CA, USA) or p62 (1:100, 7695, Cell Signaling Technology, Danvers, MA, USA) primary antibodies or isotype-matched negative controls and diaminobenzidene (Vector Laboratories Inc., Burlingame, CA, USA) as substrate. For quantitative immunohistochemistry, photomicrographs of the tumors were acquired using a Nanozoomer 2.0RS at 20X magnification (Hamamatsu, Shizuoka, Japan) and cells positive for KI67 or p62 staining in four representative areas for each tumor were counted or their intensity measured using CellProfiler.

The peptide p5+14 (with a cysteine residue at the N-terminal) was prepared for tissue staining by biotinylation, according to the manufacturer’s instructions, using a maleimide-biotin conjugation kit (Pierce, Grand Island, NY, USA). Six μm-thick formalin-fixed, paraffin-embedded human or animal amyloid-laden tissue sections were deparaffinized, placed on slides, and incubated in citrate antigen retrieval solution (Citrus Plus; BioGenex, Fremont, CA, USA) at 90 °C for 30 min. The biotinylated peptide p5+14 was added at a concentration of 5 μg/mL in PBS and incubated overnight at 4 °C in a humidified chamber. The slides were developed using the Vectastain Elite ABC development kit (Vector Labs, Burlingame, CA, USA) and visualized using diaminobenzidene (Vector Labs).
Detection of amyloid was achieved in consecutive tissue sections by staining with an alkaline Congo red solution (0.8% w/v Congo red, 0.2% w/v KOH, 80% ethanol) for 1 h at room temperature, followed by a counterstain with Mayer’s hematoxylin for 2 min. All tissue sections were examined using a Leica DM500 light microscope (Leica, Wetzlar, Germany) fitted with cross-polarizing filters (for Congo red). Digital microscopic images were acquired using a cooled CCD camera (SPOT; Diagnostic Instruments, Sterling Heights, MI, USA).