Atlantic wolffish (A. lupus)
All fish were obtained from the Montreal Biodome that maintained aquariums. Atlantic wolffish were approximately 9 to 10 years old and submitted dead on ice at the necropsy services for diagnostic purpose. Necropsy was done approximately 1 h after death, and all were well preserved. Pancreata were collected and fixed in 10% neutral buffered formalin, trimmed, and embedded in paraffin.
Routine staining, special stains, and immunohistochemistry
The paraffin sections (4 μm) were stained with hematoxylin-eosin-phloxine-saffron (HEPS) for the histologic analysis of all organs. Pancreatic sections were stained with Congo red to reveal amyloid deposits and Grimelius to identify neuroendocrine cells.
For immunohistochemistry procedure, pancreatic sections were deparaffinized and washed with PBS (pH 7.4) for 20 min. They were next permeabilized and blocked with 0.1% saponin and 3% (w/v) BSA in PBS. The tissue sections were further incubated during 1 h at 37°C. Immunofluorescence was performed with monoclonal antibodies against insulin, IAPP, glucagon, and somatostatin (Santa Cruz Biotechnology, Dallas, TX, USA). All were rabbit monoclonal antibody except for IAPP, which was a mouse monoclonal antibody. Rabbit anti-chromogranin A, anti-pancreatic polypeptide, and anti-synaptophysin antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Each antibody was applied overnight at 4°C at a 1/50 dilution with a solution containing 0.1% saponin and 3% (w/v) BSA. The pancreatic sections were washed three times with PBS containing 0.05% Tween 20™ and then incubated 1 h at 37°C in the blocking buffer containing a secondary antibody. Anti-rabbit Alexa 488 and anti-mouse Alexa 594 were used as secondary antibodies at 1/1,000 dilutions (Santa Cruz Biotechnology, Dallas, TX, USA). Negative controls were performed using the serum corresponding to the type of the secondary antibody used (mouse or rabbit). After three washes, slides were mounted with anti-fade reagent (DakoCytomation, Carpinteria, CA, USA). The cellular distribution of each pancreatic hormone was assessed using a DMI 4000B reverse fluorescence microscope. Images were captured as 8-bit tagged image format files with a DFC 490 digital camera, and the images were analyzed using the Leica Application Suite Software, version 2.4.0 (Leica Microsystems Inc., Richmond Hill, ON, Canada). Data are representative of all specimens collected.
Isolation and sequencing of Anarhichas IAPP DNA
To sequence the IAPP gene, DNA was isolated from paraffin-embedded pancreatic tissues using a DNeasy tissue kit (Qiagen, Toronto, ON, Canada). Fast-cycling PCR DNA synthesis was performed with a Techne TC-512 thermocycler with the following PCR protocol: 5 min at 96°C; 45 cycles of 5 s at 96°C, 5 s at 53°C, and 5 s at 68°C; and a final extension of 1 min at 72°C. Each PCR reaction consisted of 500 nM of IAPP primers (forward and reverse), 300 ng of DNA, and 10 μL of fast-cycling Taq DNA polymerase master mix (Qiagen, Toronto, ON, Canada). Degenerate IAPP forward (5′-AAGTGCAACACAGCCACCTG-3′) and reverse (5′-CGTTTKCCGTAGGTRTTDCGA-3′) primers were used and covered the seven first and three last amino acids of the IAPP peptide (Westermark et al. 2002). PCR products were visualized in a 2% (m/v) agarose gel with ethidium bromide, and bands of the expected size were extracted (Zymoclean™ Gel DNA Recovery Kit, Cedarlane Labs, Burlington, NC, USA). A second PCR was performed using the same primers with the following PCR protocol: 5 min at 96°C; 45 cycles of 5 s at 96°C, 5 s at 54°C, and 5 s at 68°C; and a final extension of 1 min at 72°C. Sequencing was performed using the Mcgill gene sequencing platform. The amino acid sequence of Anarhichas IAPP was deducted based on DNA sequence (http://didac.free.fr/seq/dna2pro.htm).
In silico analysis of fibril formation
The tendency for β-sheet aggregation of each amino acid sequence was calculated based on the Agg parameter, obtained using in silico analysis with the TANGO program (http://tango.crg.es), which predicts protein aggregation. A higher Agg score is indicative of a peptide that can form potentially toxic aggregates. The amino acid sequence in the 19-34 segment of Atlantic salmon (Salmo salar), daddy sculpin (M. scorpius), and zebrafish (Danio rerio) IAPP previously published were selected to compare the Agg parameter (Westermark et al. 2002). Human and feline IAPP amino acid sequences were used as positive controls, and rodent IAPP was representative of a negative control. Control sequences were obtained from GenBank (www.ncbi.nlm.nih.gov/Genbank), with the following accession numbers: Homo sapiens [GenBank: M26650.1], Felis catus [GenBank: NM001043338.1], Rattus norvegicus [GenBank: NM012586.2].