Carl Zeiss, rotatable analyzer +/25u, 6625, rotatable compen- sator Lambda, +/25u, 6625). Generation of polyclonal antisera The presumed mature sequences spanning residues 24–580 of zebrafish (Danio rerio) PrP-1 and residues 18–539 of zebrafish PrP-2 (sequence data provided by Dr. Edward Ma´laga-Trillo, Depart- ment of Biology, University of Konstanz), were each amplified from genomic DNA, whereas the mature sequence of gilthead sea bream (Sparus aurata) PrP-1 spanning residues 26–475 was cloned from plasmid DNA. All three were cloned into the pET21d DNA vector (Novagen, San Diego, CA) to produce recombinant proteins tagged with six histidine residues at the C-terminus. After sequence verification by double-stranded sequencing, the recombinant proteins were expressed in BL21 (DE3) E.coli (Stratagene, La Jolla, CA) with IPTG induction from single clone colonies. The recombinant proteins were purified under denatur- ing conditions from cell lysates on Ni-NTA agarose columns (Qiagen, Hilden, Germany) and then specifically eluted with imidazole. The polyclonal antisera ZebPrP1, ZebPrP2 and SaurPrP1 were raised against the purified zebrafish PrP-1, zebrafish PrP-2 and sea bream PrP-1 proteins respectively, by 3 successive subcutaneous inoculations of rabbits with 150 to 200 mg of recombinant protein at 4 weeks intervals. All pertinent sequence data are deposited in GenBank and the accession numbers are given at the end of the manuscript. Affinity purification of SaurPrP1 polyclonal antiserum 2 aliquots containing 150 mg of gilthead sea bream recombinant PrP-1 protein each, were loaded onto 10% polyacrylamide gels and after SDS-PAGE, they were each electrotransferred to a nitrocellulose or a PVDF membrane. After electrophoresis the two membranes were stained with amido black staining solution (0.1%) and the protein-containing membrane pieces were finally excised. After a blocking step in 50 mM Tris HCl [pH 7.4], 150 mM NaCl, 0.05% Tween 20 (TBST) containing 5% milk, for 1 hr at RT, each membrane piece was probed with blocking buffer containing 500 ml of SaurPrP1 antiserum, at 4uC overnight. Following several washes, the IgGs that specifically bound to PrP- 1, were finally eluted from the membranes with 0.2 M Glycine.HCl [pH 2.5], for 5 min at 4uC. Each eluate was neutralized with 2 M Tris HCl [pH 9.0], and then dialyzed overnight at 4uC in 50 mM Tris HCl [pH 7.4], 150 mM NaCl (TBS), using 100 mm-Spectra/Por molecularporous membrane tubing (Spectrum Medical Industries, Los Angeles, USA). Following dialysis, the purified IgGs were saturated in buffer containing 20 mM Tris HCl [pH 8.4], 150 mM NaCl, 5 mM EDTA, 1% gelatin, 0.1% BSA. Depletion of recombinant gilthead sea bream PrP-1 – specific immunoglobulin fraction from SaurPrP1 polyclonal antiserum SaurPrP1 polyclonal antibody was diluted in phosphate buffered saline (1:2000), containing 5% normal goat serum, 2.5% BSA and 0.05% Tween 20. The antibody was incubated with 0.6 mM of recombinant gilthead sea bream PrP-1 protein at 4uC overnight. The depleted antiserum was briefly centrifuged before use in all negative immunohistochemistry control experi- ments. Immunohistochemistry Four different polyclonal anti-PrP antibodies were used for the immunohistochemical detection of the endogenous PrP proteins of TSE Transmission to Sea Bream sea bream, namely ZebPrP1 (1:1000), ZebPrP2 (1:1000), SaurPrP1 (1:2000) and FuguPrP1 (1:500), the latter being raised by our group against PrP-1 protein of Takifugu rubripes. The commercially available monoclonal antibody, 12F10 (Cayman Chemical, Ann Arbor, MI), raised against amino acids 142–160 of human PrP, was used for the detection of residual mammalian PrP (1:200), since it also displays cross-reactivity with both ovine and bovine PrP. All paraffin sections were cut at 4 mm thickness. Depending on the prion protein of interest, PrPC or PrPSc, two different pretreatment protocols were used. For PrPC labeling, an antigen retrieval step was performed by boiling in citrate-buffered saline [pH 6.0] for 7 minutes before the staining procedure. For PrPSc detection, the sections were hydrated-autoclaved at 121uC for 30 minutes, then incubated for 5 minutes in 90% formic acid prior to an 8 minutes-incubation with proteinase K (Dako, Glostrup, Denmark) at RT. Sections were treated with appropriate biotinylated secondary antibodies (Vector Laboratories, Burlin- game, CA) and visualized using the avidin-biotin method-based Vectastain Elite ABC and the Diaminobezidine substrate kits (Vector Laboratories, Burlingame, CA) according to the manu- facturer’s instructions. Negative controls for immunohistochemis- try involved omitting the primary antibody. Staining with polyclonal (anti 14-3-3b, Santa Cruz, California, USA) and monoclonal antibodies (12F10, 6H4) raised against proteins of mammalian origin was also performed. The mouse anti-tubulin monoclonal antibody (Abcam, Cambridge, UK) and the mono- clonal SAF84 antibody (raised against SAF preparation from infected hamster brain, assumed epitope 126–164) were used as well. PrPSc enrichment and Western blot analysis Western blot analysis of potentially enriched mammalian and teleost PrPSc was performed on brain homogenates from BSE- infected cows, scrapie-infected sheep, and TSE-challenged fish. Briefly, aliquots of 10% (w/v) brain homogenate were digested for 1 hr at 37uC with proteinase K at 25 mg/ml for sheep, 30 mg/ml for cow and 0.1–10 mg/ml for sea bream. PMSF (5 mM) was added to stop the protease digestion and PrPSc was precipitated with NaCl (10%) (w/v). The pellet was washed with 25 mM Tris HCl [pH 8.8] containing 0.05% sarkosyl and then resuspended in an appropriate volume of 2.56 O’Farrell buffer for gel electrophoresis. For western blot analysis, untreated and proteinase K treated brain homogenates were analyzed by SDS-PAGE on 12% polyacrylamide gels and the separated proteins were then transferred onto PVDF membranes. After blocking with phos- phate buffered saline containing 0.1% Tween 20 (PBST) and 5% milk, the immunoblots were probed with the fish-PrP specific polyclonal antisera, ZebPrP1 (1:10000), ZebPrP2 (1:35000), SaurPrP1 (1:20000), FuguPrP1 (1:20000), and the monoclonal antibody 6H4 (1:5000) (Prionics, Zurich, Switzerland) overnight at 4uC. After washing, they were incubated for 1 hr with either alkaline-phosphatase or horseradish-peroxidase conjugated sec- ondary goat anti-rabbit or anti-mouse antibodies (Pierce, Rock- ford, IL) diluted 1:10000 in PBST. The blots were developed using the CDP-Star chemiluminescent substrate (NE Biolabs, Beverly, MA), or the ECL Western blotting Substrate (Pierce, Rockford, IL), depending on the secondary antibody and according to the manufacturer’s instructions. GenBank Accession Numbers Danio rerio prion protein 1 coding sequuence: AY438683 Danio rerio prion protein 1: AAS00159 Danio rerio prion protein 2 coding sequence: AY438684 PLoS ONE | www.plosone.org 11 July 2009 | Volume 4 | Issue 7 | e6175