Synthetic poly(A), catalog number P9403 (0.2–6 kb by agarose gel electrophoresis), was purchased from Sigma. Synthetic 100-mer oligo(dA), -(dT), -(dC), and -(dG), 100-mer fluorescein-labeled oligo(dT) and oligo(dA), and 100-mer biotin-labeled oligo(dT) were purchased from Operon (Huntsville, AL). Branched oligo(dT) constructs were purchased from Fidelity Systems (Gaithersburg, MD). EasyTides [α-32P]ATP (catalog number BLU503H250UC) was obtained from PerkinElmer Life Sciences. Stock solutions of synthetic polynucleotides were dissolved in 1× TE, pH 8.0, and concentrations were confirmed by A260 prior to use. Dynabeads® M-450 rat anti-mouse IgM (catalog number 110-39D) and Pro-Long® Antifade (catalog number P7481) were obtained from Invitrogen. The anti-PrP 27–33 mAb was obtained from Dr. Peter Morganelli (VA Hospital, White River Junction, VT). The 15B3 mAb, 15B3 coating buffer, immunoprecipitation buffer, and IP wash buffer were obtained from Prionics AG (Schleiren-Zurich, Switzerland). The 89–112 and D13 mAbs were kindly provided by Dr. Anthony Williamson (La Jolla, CA). Benzonase® (catalog number 70664-3) was obtained from Novagen (Madison, WI). The bacterially expressed recombinant mouse PrP was kindly provided by Roland Riek (La Jolla, CA). Anti-actin antibody (catalog number A5060), polyadenylic acid-Sepharose (catalog number P2765), heparin-agarose (catalog number H3025), poly-l-glutamate (catalog number P4886), heparan sulfate (catalog number H7640), polynucleotide phosphorylase (catalog number P2869), acetylated bovine serum albumin (catalog number B8894), hexokinase (catalog number H4502), SP-agarose (catalog number S1799), deoxycholic acid, sodium salt (catalog number D6750), MOPS (catalog number M5789), MES (catalog number M5287), and imidazole (catalog number I-5513) were purchased from Sigma. CNBr-activated Sepharose 4 Fast Flow (catalog number 17-0981-01) was purchased from Amersham Biosciences. RNase-free water and stock solutions of NaCl, EDTA, TE, pH 8.0, and Tris, pH 8.0, were purchased from Ambion (Austin, TX). Other chemical reagents, including protein A-agarose (catalog number PI20333) were purchased at the highest grade available from Fisher. All phosphate-buffered saline (PBS) was without calcium or magnesium and purchased from Mediatech, Inc. (Herndon, VA). Teflon-printed slides (catalog number 63415-15) were obtained from Electron Microscopy Sciences (Hatfield, PA). The hamster scrapie strain Sc237 was kindly provided by Dr. Stanley Prusiner (San Francisco). Sc237 was originally isolated in inbred Syrian hamsters by Richard Marsh (Madison, WI) and subsequently passaged in outbred Syrian hamsters.
Preparation of PrPC Substrate
All procedures were performed at 4 °C. Six frozen brains from 8- to 12-week-old golden Syrian hamsters of either sex (Harlan Bioproducts, Indianapolis, IN) were suspended in ice-cold PBS plus Complete™ protease inhibitors (Roche Applied Science) to 40 ml total volume and homogenized using a Biospec Products (Bartlesville, OK) Biohomogenizer Mixer at 7000 rpm for 30–60 s. The homogenate was centrifuged at 3200 × g for 20 min, and the pellet was resuspended in a total volume of 40 ml of PBS, 1% deoxycholate, 1% Triton X-100, and Complete™ protease inhibitors using a Kontes (Vineland, NJ) glass Dounce homogenizer (10 strokes with pestle B). The homogenate was solubilized by incubation on ice for 30 min and then centrifuged at 100,000 × g for 30 min. The supernatant was filtered using Millipore (Billerica, MA) 0.2-µm Stericups and poured over a 1-ml ImmunoPure immobilized protein A column (Pierce) to pre-clear tissue-derived immunoglobulins. The flow-through was collected and passed over a Econ-Pac column (Bio-Rad) packed with 1 ml of protein A-agarose resin (Pierce) cross-linked to 3F4 antibody (Signet Laboratories, Dedham, MA) using DMP (Pierce). The column was washed with 15 ml of 20 mm Tris, pH 8.0, 0.5 m NaCl, 5 mm EDTA followed by 10 ml of PBS, 0.5% Triton X-100, and then eluted with 7.2 ml 0.2 m glycine, pH 2.5. The eluate was combined with 800 µl of 1 m Tris, pH 9.0 and applied to Zeba (Pierce) desalting spin columns pre-equilibrated in 20 mm MES, pH 6.4, 0.15 m NaCl, 1% Triton X-100. The resulting 8-ml buffer-exchanged sample was then passed over a 1.5-ml SP-Sepharose ion exchange column (Sigma), and the column was washed with 15 ml of 20 mm MOPS, pH 7.0, 0.25 m NaCl, 1% Triton X-100. The column was eluted with 8 ml of 20 mm MOPS, pH 7.0, 0.5 m NaCl, 1% Triton X-100 and applied to Zeba desalting spin columns pre-equilibrated with 20 mm MOPS, pH 7.5, 0.15 m NaCl, 0.5% Triton X-100 to reduce the salt concentration of the final product. The typical yield from a preparation using six hamster brains is ~ 16 µg of PrPC (~2 µg/ml), based on comparison with known concentrations of Escherichia coli-expressed recombinant PrP, using semi-quantitative Western blots and silver stain gels.
PMCA and serial dilution/propagation experiments were performed as described by Castilla et al.
), using purified substrates instead of brain homogenates. To avoid cross-contamination, all work was carried out in laminar flow bio-safety cabinets using disposable surfaces and aerosol barrier tips. Typically, we prepared a mixture containing 200 µl of PrPC
substrate diluted in 20 mm
MOPS, pH 7.5, 0.15 m
NaCl, 0.5% Triton X-100 (note that the appropriate dilution of PrPC
substrate is empirically determined for each individual preparation using seeded, serial PrPSc
propagation assays; the final concentration of PrPC
in the mixture is typically between 250 and 500 ng/ml), 40 µl of imidazole diluted in 20 mm
MOPS, 0.15 m
NaCl, 0.5% Triton, pH 7.0, 4 µl of 0.5 m
EDTA, pH 8.0, and 116 µl of TE buffer. From this mixture, multiple 90-µl aliquots were dispensed into 0.5-ml thin walled PCR tubes. Where indicated, the TE buffer contained a polyanionic compound such as poly(A) RNA or synthetic oligonucleotides at a concentration of 68.96 µg/ml (to produce a final concentration of 20 µg/ml in the reaction) except for polyglutamate, heparan sulfate, oligo(dT) construct 1, oligo(dT) construct 2, and oligo(dT) construct 3, which were used at final concentrations of 2 and 50 µg/ml and 800, 80, and 80 nm
, respectively. Aliquots were then frozen at −70 °C until use in PMCA reactions. Serial propagation reactions were initiated on day 0 by the addition of 10 µl of PrPSc
or PrP27–30 of PrP (or 10 µl of PrPC
dilution buffer (20 mm
MOPS, pH 7.5, 0.15 m
NaCl, 0.5% Triton X-100) for unseeded reactions) to both day 0 and day 1 tubes. Day 0 samples were immediately refrozen without sonication, and day 1 samples were subjected to PMCA for 24 h using a Misonix 3000-MPD programmable sonicator equipped with a microplate horn containing 350 ml of water, set for 30-s bursts every 30 min at output ≤6.0. The appropriate output setting was determined empirically for each individual horn and changed with usage. Temperature was maintained by circulating heated water through a section of aluminum coil to heat the air inside the acoustic chamber (Misonix, Farmingdale, NY), and the microplate horn was covered with a sheet of plastic film to minimize evaporation. Before each burst, the bath temperature measured 41 °C, and after each burst the temperature measured 42 °C. Samples were mounted into a custom-made Plexiglas holder with the lid designed to keep the bottom of the PCR tubes ~3 mm from the horn surface, and the tubes were tightly closed. Samples were generally mounted no further than 4 cm from the center of the circular horn. With appropriate sonicator settings, the temperature inside a thin walled PCR tube filled with 250 µl of water subjected to a single 30-s pulse typically rose 1–3 °C (measured by thermistor readings). Immediately following the last sonication cycle of the first 24-h round, the day 1 PMCA product was removed from the sonicator and centrifuged for 5 s. Pipetting up and down several times resuspended the sample, and 10 µl was used to seed the thawed day 2 aliquot containing a fresh substrate mixture. The remainder of the day 1 sample was stored at −70 °C until analysis, and the day 2 sample was subjected to PMCA for 24 h in the second round of propagation. This process was repeated for multiple propagation rounds, up to 4 days.
PrPSc Detection Assays
All protease-digested (+PK) samples were incubated with 50 µg/ml proteinase K (Roche Applied Science) for 1 h at 37 °C. An equal volume of 2× SDS sample buffer was then added, and samples were boiled for 10 min at 95 °C. SDS-PAGE was performed on 1.5-mm 12% polyacrylamide gels with an acrylamide:bisacrylamide ratio of 29:1 (Bio-Rad). Following electrophoresis, the proteins were transferred to a methanol-charged, buffer-equilibrated polyvinylidene difluoride membrane (Millipore) using a Trans-blot SD semi-dry transfer cell (Bio-Rad) set at 2 mA/cm2 for 30 min.
To visualize PrP signals, Western blot membranes were first treated with 3 m GdnSCN (Roche Applied Science) at room temperature for 30 min. The membranes were then rinsed with TBST (10 mm Tris, pH 7.2, 150 mm NaCl, 0.1% Tween 20) and blocked for 1 h in Hood skim milk (Chelsea, MA) buffered with TBST. The blocked membrane was incubated overnight at 4°C with 3F4, 6D11 or 27–33 mAbs (Signet) diluted 1:5000 or 1:10,000, respectively, in TBST. Following this incubation, the membrane was washed three times for 10 min in TBST and incubated for 1 h at 4 °C with horseradish peroxidase-labeled anti-mouse IgG secondary antibody conjugate (GE Healthcare) diluted 1:5000 in TBST. The membrane was washed again four times for 10 min with TBST. The blot was developed using West Pico or West Femto (Pierce) chemiluminescence substrate, sealed in plastic covers, and either exposed to Fujifilm (Tokyo, Japan) Super RX film or captured digitally using a Fuji (Fujifilm) LAS-3000 chemiluminescence documentation system. Exposed films were developed automatically in a Kodak M35A X-Omat film processor, and digital images were captured using Image Reader version 2.0 and analyzed with Image Gauge version 4.22 software (Fujifilm, Tokyo, Japan). Relative molecular masses were based on migration of pre-stained standards from either Fermentas (Hanover, MD) or Bio-Rad.
Immobilized Polyanion-PrP Binding Assays
Crude hamster and mouse brain homogenates were prepared using a Teflon/glass Potter homogenizer. Brains were homogenized in cold PBS plus Complete™ protease inhibitor mixture to a final brain concentration of 10% (w/v). Crude homogenates were centrifuged for 30 s at 200 × g, and the post-nuclear supernatants were diluted in PBS plus 1% n-octyl β-d-glycopyranoside, 5 mm EDTA to a 1% final brain concentration. Supernatants were solubilized by incubating at 4 °C for 1 h and then centrifuged for 30 min at 20,800 × g at 4°C. 300 µl of solubilized supernatants were incubated with 50 µl of packed polyadenylic acid-Sepharose, heparin-agarose, or polyglutamate-Sepharose (coupled to CNBr-activated Sepharose according to the manufacturer’s instructions) overnight at 4 °C with tilt rotation. Following incubation, the samples were subjected to brief centrifugation, and the sample supernatants were removed. The Sepharose/agarose pellets were washed with PBS plus 1% n-octyl β-d-glycopyranoside, 5 mm EDTA. Pellet-associated proteins were eluted by boiling the pellet with 50 µl of SDS loading buffer. Samples were subjected to SDS-PAGE and Western blot detection with anti-PrP mAb 27–33. Subsequently, each blot was stripped in 3 m guanidine isothiocyanate, 20 mm Tris, pH 8.0, for 1 h at room temperature and re-probed with anti-actin antibody.
PrPC Solubility Assay
Seeded and unseeded 100-µl PMCA reactions were performed in duplicate for each condition. Before addition to each sample, the PrPC substrate was subjected to 100,000 × g centrifugation for 45 min at 4 °C, and the supernatant fraction was then used as the source of PrPC for each sample. After one round of PMCA, 1 ml of PBS, 0.5% Triton X-100 was added to all samples that were then subjected to 100,000 × g centrifugation for 1 h at 4 °C. The sample supernatants were removed; the pellets were washed with 1 ml of PBS, 0.5% Triton X-100, and the centrifugation step was repeated. All but 100 µl of the sample supernatants were removed by aspiration, and the pellets were resuspended by vortexing. An equal volume of 2× SDS loading buffer was added, and each sample was boiled for 10 min at 95 °C. Samples containing the input amounts of PrPC and PrPSc added to the experimental samples were prepared at the same time, and all samples were analyzed together by Western blot with 3F4 mAb.
89–112 mAb PrP Immunoprecipitation
100-µl PMCA reactions were carried out in duplicate for each condition. Seeded reactions contained PrPSc template generated in a serial PMCA propagation reaction stimulated with synthetic poly(A) RNA. After overnight PMCA, 400 µl of IP buffer (TBS, 3% Tween 20, 3% Nonidet P-40) was added to each sample. 1.5 µg of 89–112 mAb was added to each sample for a final concentration of 3 µg/ml, and the samples were incubated for 2 h at room temperature with slow tilt rotation. 60 µl of a 50% slurry of Immunopure Protein A Plus (Pierce) was added to each sample and allowed to incubate for 40 min at room temperature with slow tilt rotation. Each sample was then centrifuged for 30 s, and the supernatant was removed by aspiration. 750 µl of wash buffer (TBS, 2% Tween 20, 2% Nonidet P-40) was added to each sample that was then mixed briefly by vortexing. The samples were centrifuged again for 30 s, and the wash buffer was removed by aspiration. Each sample was washed three more times in the same manner followed by a final wash with PBS. After aspiration of the PBS, 50 µl of 1× SDS loading buffer was added, and each sample was boiled for 10 min at 95 °C. All samples were analyzed together by Western blot with 3F4 mAb.
15B3 mAb PrP Immunoprecipitation
Rat anti-mouse IgM Dynabeads® were coated with 15B3 mAb at a concentration of 2 µg of antibody per 100 µl of beads. Beads were incubated with 15B3 in coating buffer for 2 h at room temperature with slow tilt rotation. 15B3-coated beads were washed three times with coating buffer and then resuspended in a volume of coating buffer equivalent to the starting volume of beads. A Dynal MPC™-S Magnetic Particle Concentrator (Invitrogen) was used for all steps involved in 15B3 bead preparation and PrP immunoprecipitation.
For PrP immunoprecipitation, 100 µl of PMCA reactions were carried out for each condition. Seeded samples contained PrPSc template generated in a serial PMCA propagation reaction stimulated with synthetic poly(A) RNA. Each sample was combined with 400 µl of IP buffer and 15 µl of 15B3-coated beads. Samples were immunoprecipitated for 2 h at room temperature with slow tilt rotation. The beads in each sample were washed three times with 1 ml of wash buffer. 30 µl of 2× SDS loading buffer was added to the beads in each sample, which were then boiled for 5 min at 95 °C. All samples were analyzed together by Western blot with 6D11 mAb.
Fluorescence Microscopy Analysis of Purified PrP and Fluorescein-(dT)100
For each sample, a seeded or unseeded 200-µl PMCA reaction was carried out. The polyanion added to the reactions was a synthetic (dT)100 or (dA)100 labeled with a fluorescein group at the 5′ end of the oligonucleotide. The stock oligonucleotide was prepared in 1× TE, pH 8.0, and added to each reaction at a final concentration of 20 µg/ml. After one round of PMCA, each sample was combined with 800 µl of PBS and subjected to 100,000 × g centrifugation for 45 min at 4 °C. All but ~100 µl of the sample supernatants was removed. At this point, MgCl2 was added to a final concentration of 2 mm along with 50 units of Benzonase to those samples undergoing nuclease digestion. Benzonase -treated samples were then incubated for 30 min at 37 °C with shaking at 300 rpm followed by addition of EDTA to a final concentration of 15 mm to inactivate the Benzonase. Following the first centrifugation step, and the incubation step for Benzonase-treated samples, all samples were resuspended in PBS up to a final volume of 1 ml and subjected to 100,000 × g centrifugation for 45 min at 4 °C. All but ~ 150 µl of the sample supernatants were removed. Samples treated with Benzonase were subjected to an additional centrifugation and wash step. The sample pellets were then resuspended by vortexing and applied to 15-mm, single well, Teflon-printed slides that were pre-coated with gelatin subbing solution. To prepare gelatin subbing solution, 6 g of 300 bloom gelatin was dissolved at 58 °C in 500 ml of sterile distilled water. 0.2 g of chromium potassium sulfate was added to the solution after the gelatin dissolved. Gelatin subbing solution cooled to 50 °C was applied to the slides in a volume large enough to cover the entire well and then allowed to dry for at least 2 h at room temperature. Samples were incubated on the slides overnight in a humidified chamber at 4 °C. The following day, the samples were fixed to the slide by addition of 150 µl of PBS, 8% formaldehyde. The samples were allowed to incubate at room temperature for 15 min and then removed by aspiration. Each slide well was washed one time with 150 µl of PBS. 150 µl of mAb D13 diluted 1:250 in PBS + 4% BSA was applied to each slide well and allowed to incubate overnight at 4 °C or for 2 h at room temperature in a humidified chamber. After primary antibody incubation, the slide well was washed three times with 150 µl of PBS + 4% BSA and allowed to incubate for 15 min at room temperature during each wash. 150 µl of secondary antibody, sheep anti-mouse conjugated with Alexa Fluor 568 (Invitrogen), diluted 1:250 in PBS + 4% BSA, was applied to each slide and allowed to incubate for 2 h in the dark at room temperature in a humidified chamber. The slide well was then washed three times with 150 µl of PBS + 4% BSA and allowed to incubate for 15 min at room temperature during each wash. 15 µl of ProLong® antifade solution was added to each sample and number 1.5, 18-mm square glass coverslips (Corning Glass) were mounted on each slide and allowed to dry overnight in the dark in a desiccating chamber. We also prepared and analyzed a control slide coated with only reaction buffer and then stained with D13 mAb and Alexa Fluor 568 secondary antibody to rule out nonspecific binding of either antibody to the slide. A slide coated with only fluorescein-labeled (dT)100 in reaction buffer was prepared to rule out nonspecific binding of the fluorescein-labeled (dT)100 to the slide. Finally, a slide coated with PrPC in reaction buffer that did not contain fluorescein-labeled (dT)100 was prepared to rule out autofluorescence of PrPC in the fluorescein excitation wavelength. Samples were analyzed by fluorescence microscopy using a Leica Confocal model TES SP confocal scanning microscope (Leica Microsystems, Heidelberg, Germany). All images were captured digitally using a 63×, 1.32 aperture, oil immersion objective lens, and Leica confocal system software, version 2.61. Image resolutions are 1024 × 1024 and are averages of three consecutive scans. Alexa Fluor 568 is displayed as magenta to be red-green color-blind-compatible. Images were processed (cropped and re-sized) using Photoshop software.
Enzymatic Synthesis of [32P]Poly(A) RNA
Radiolabeled poly(A) RNA was synthesized in a 100-µl reaction containing 100 mm Tris-HCl, pH 9.0, 0.4 mm EDTA, pH 7.0, 5 mm MgCl2, 6 mm ATP, 70 mm glucose, 0.1 mg/ml acetylated bovine serum albumin, 2.5 units/ml polynucleotide phosphorylase, 20 units/ml hexokinase, and 2 mCi/ml of 3000 Ci/mmol [α-32P]ATP. All components of the reaction, except polynucleotide phosphorylase, hexokinase, and [α-32P]ATP, were combined and pre-warmed to 37 °C. The enzymes and [α-32P]ATP were then added, and the reaction mixture was incubated at 37 °C for 45 min. Following incubation, the synthesized poly(A) RNA was purified using an RNeasy kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. The concentration of the purified poly(A) RNA was determined by measuring A260
Nuclease Protection Assay
For each condition 300-µl PMCA reactions were prepared on ice. Samples without PrPC contained PrPC dilution buffer (20 mm MOPS, pH 7.5, 0.15 m NaCl, 0.5% Triton X-100) in the place of PrPC. [32P]Poly(A) RNA diluted into 1× TE, pH 8.0, was added to each sample for a final concentration of 20 µg/ml. Samples not subjected to PMCA were frozen at −70 °C. The remaining samples were subjected to PMCA for 24 h. After 24 h the samples subjected to PMCA were frozen at −70 °C for 30 min and then all samples were thawed on ice. 700 µl of PBS, 0.5% Triton X-100 was added to each sample, which was then mixed briefly by vortexing and subjected to 100,000 × g centrifugation for 45 min at 4 °C. All but ~50 µl of the sample supernatants were removed. The sample pellets were washed by addition of 950 µl of PBS, 0.5% Triton X-100, briefly mixed by vortexing, followed by 100,000 × g centrifugation for 45 min at 4 °C. All but ~50 µl of the sample supernatants were removed. 100 µl of Benzonase digestion buffer (50 mm Tris-HCl, pH 9.0, 20 mm NaCl, 2 mm MgCl2) was added to each sample, and the pellets were resuspended by pipetting. 2 µl of 25 units/µl Benzonase was added to nuclease-treated samples. All samples were then incubated at 37 °C for 45 min with 300 rpm shaking (Eppendorf Thermo-mixer, Fisher). 50 µl of 100 mm EDTA, pH 8.0, and 900 µl of PBS, 0.5% Triton X-100 were added to all samples. Each sample was then mixed briefly by vortexing and subjected to 100,000 × g centrifugation for 45 min at 4 °C. All but ~50 µl of the sample supernatants were removed, and 950 µl of PBS, 0.5% Triton X-100 was added to each sample pellet. Each sample was then mixed briefly by vortexing, and subjected to 100,000 × g centrifugation for 45 min at 4 °C. All but ~20 µl of the sample supernatants were removed. An equal volume of denaturation buffer (8 m urea, 10 mm Tris phosphate, pH 8.0, 0.5% Triton X-100) and 2 µl of 10× loading buffer (0.25% bromphenol blue, 50% glycerol) was added to each sample. An input control [32P]poly(A) RNA sample was prepared at the same time, and all samples were heated at 70 °C for 10 min. Denatured samples were subjected to electrophoresis on a 1.2% 1× TBE-agarose gel and then transferred to Biodyne B nylon membrane (Pierce) using 20× SSC transfer buffer and Whatman 3MM chromatography paper (Fisher). After overnight transfer (~17 h) the membrane was exposed to Biomax MS film (Eastman Kodak Co.) with a Hyperscreen intensifier screen (Amersham Bio-sciences). To calculate the stoichiometry of the interaction between nuclease-protected [32P]poly(A) RNA and PrPC, we incubated [32P]poly(A) RNA with PrPC at 37 °C overnight. The sample was then subjected to centrifugation, and the detergent-insoluble fraction was treated with Benzonase. The sample was washed and centrifuged again to re-isolate the insoluble fraction. This fraction was denatured and subjected to agarose gel electrophoresis, transferred to a nylon membrane, and exposed to x-ray film.
Preparation of Nuclease Pre-treated PrPSc
derived from the fourth round of a seeded PMCA propagation reaction was diluted 1:10 into PBS, 2% Triton X-100. Brain-derived 237 PrP27–30 was prepared as described previously and also diluted 1:10 into PBS, 2% Triton X-100 (9
). 20 µl of each PrPSc
dilution was combined with 180 µl of PBS, 2% Triton X-100, 4 µl of 100 mm
, and either 25 or 10 µl of 25 units/µl Benzonase was added to the PrP27–30 preparation and propagated PrPSc
samples, respectively. Identical mock-digested samples were prepared except PBS was added in the place of Benzonase. Samples were incubated at 37 °C for 30 min with shaking at 300 rpm. EDTA was added to each sample to a final concentration of 15 mm
to inactivate the Benzonase, and samples were subjected to 100,000 × g
centrifugation for 1 h at 4 °C. After centrifugation, all but ~15 µl of the sample supernatants were removed by aspiration. Sample pellets were resuspended in 185 µl of PBS, 2% Triton X-100 for a final volume of 200 µl and then sonicated for 30 s in a Misonix 3000-MPD programmable sonicator equipped with a microplate horn containing 350 ml of water and set for output ≤6.0. 10 µl of each PrPSc
preparation was used to seed 100-µl PMCA propagation reactions.
In Situ PrP Immunohistochemistry and Acridine Orange Histochemistry
To study in situ
co-localization of PrP and polyanions, scrapie-infected (n
= 3) and wild type (n
= 3) hamster brains were fixed in 10% formalin. Tissue sections were pretreated for antigen retrieval using established protocols (75
). Briefly, tissue sections were deparaffinized in xylene followed by a 100, 95, 75, and 50% ethanol series. After a water rinse, sections underwent the following: 1) hydrated autoclaving at 121 °C for 10 min; 2) microwave boiling in 10 mm
citric acid buffer, pH 6.0, for 7 min; and 3) 88% formic acid for 7 min. Tissue sections stained with acridine orange were first washed with PBS and then stained with PBS, pH 6.0, 0.1% acridine orange (Molecular Probes, Invitrogen) for 15 min, differentiated in 0.1 m
, and washed with water. For PrP immuno-histochemistry, tissue sections were blocked with 10% normal goat serum in PBS for 30 min at room temperature and incubated overnight at 4 °C with a 1:50 dilution of 3F4 anti-PrP primary mouse IgG (Covance Research Products, Inc). Slides were then washed with PBS and incubated for 2 h at 37 °C with a 1:200 dilution of green fluorescent Alexa Fluor 488-labeled secondary anti-mouse IgG (Molecular Probes, Invitrogen). Samples digested with RNase (Roche Applied Science) were incubated for 1 h at 37 °C with 600 µg/ml of RNase in PBS and then washed with PBS. Tissue section slides treated with heparinase III (Sigma) were digested with 300 µl/slide of 1.6 units/ml heparinase in PBS, 4 mm
for 4.5 h at room temperature and then washed with PBS. RNase and heparinase treatment preceded immunohistochemistry, followed by acridine orange staining was performed last. After processing, all tissue sections were air-dried and mounted in Vectashield (Vector Laboratories) with 5 µg/ml 4′,6-diamidino-2-phenyl-indole (Sigma). Images were acquired with a Zeiss Axiophot fluorescence microscope with ×20 air or ×100 oil lenses and Texas Red and fluorescein isothiocyanate filter sets (Chroma). Images were captured with IPlab with manual control, equal and corresponding exposures, and adjusted and merged with ImageJ. Alexa Fluor 488 staining is pseudo-colored green, and acridine orange staining is displayed as magenta to be red-green color-blind-compatible.
Benzonase Activity Test
To test for Benzonase activity in PMCA reactions containing oligo(dT), 50 ng of biotin-labeled (dT)100
was digested with Benzonase under the same conditions as used in . Control samples containing biotin-labeled (dT)100
not treated with Benzonase were also prepared. After treatment, all samples were transferred by slot blot to Biodyne B membrane (Pierce) pre-soaked in 6× SSC. The membrane was processed using the North2South chemiluminescent detection kit (Pierce) with a streptavidin-horseradish peroxidase secondary antibody. To test for Benzonase activity in PMCA reactions containing RNA, 50 ng (~2000 cpm) of synthetic [32
P]poly(A) RNA was digested with Benzonase under the same conditions as used in and supplemental Fig. S4A
. A control sample also containing 50 ng (~2000 cpm) of synthetic [32
P]poly(A) RNA was prepared but not digested with Benzonase. After treatment, all samples were transferred by slot blot to Biodyne B membrane (Pierce) pre-soaked in 20× SSC. The membrane was exposed to Kodak BioMax MS film.
Fluorescein (dT)100-PrP co-localization assay