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assess any spongtform change by htstopathologtcal exammatron. If the brain at
necropsy 1s considered to be adequately preserved, then the sites spectfied
below should be sampled for SAF or PrPSC detection. However, If the brain IS
determmed to be autolysed at necropsy and, m the opmton of the pathologrst, IS
unsuitable for htstopathology, it IS recommended that the bramstem regions be
sampled, mcludmg the medulla region usually retamed for htstopathological
The source tissue for both SAF detection by TEM and PrPSC by Western
tmmunoblottmg must be untreated and especially not fixed m formalm or other
fixatrve Ideally rt should be processed as soon after death as practical but some
autolysts will not mvahdate the examinatton (22,231. It 1s recommended that tf
a srgmticant delay 1s likely between death and the mtttatron of extraction of
SAF and PrPSC (e.g., more than 12 h) then samples of brain and sputa1 cord
should be frozen, ideally at -70°, but -20°C is acceptable It has been recom-
mended that tissue from more than one CNS site be sampled, parttcularly for
natural scrapte cases (24) Samples for processmg should maximize the gray
matter content by drssectmg off whrte matter. Thus, for spinal cord only the
central core would be used and for tissue from cerebral hemispheres and cer-
ebellum only gray matter should be used for the extracttons If BSE brain 1s
autolysed and unsuitable for htstopathologrcal exammation, the basal nuclei
and mtdbram can be used as additional sites (2.5)
2.1. I. Tissue Requirements
for Electron Microscopy (EM) and lmmunoblotting (/MB)
1 Scrapie (sheep, goats, and moufflon) For both fresh and autolysed tissue the
requrrement IS 1 g (EM), 4 g (IMB) of spmal cord (cervical vertebrae segments
Cl and C2) plus 1 g (EM), 4 g (IMB) of bramstem region
2 BSE (cattle or other bovrdae) For fresh tissue the requirement IS 1 g (EM), 4 g
(IMB) of spinal cord (cervical vetebrae segments Cl and C2) plus 1 g (EM), 4 g
(IMB) of caudal medulla, avordmg damage to the obex
3 For autolysed BSE tissue the requirement IS™ 1 g (EM), 4 g (IMB) of bramstem,
to Include the medulla at obex
These are the mmtmum tissue requirements for one test but collectmg larger
quantities does enable retests to be conducted tf necessary
Diagnosis of BSE and Scrap/e 89
2.1.2. Reagents Used m the Methods Described
Solutton A (For detergent homogenization) 10 g N-lauroylsarcosme, sodmm salt
(Sigma [Poole, Dorset, UK] cat no L.5 125) in 100 mL of sterile deionized water
Adjust to pH 7 4 with 1A4sodium dihydrogen orthophosphate Store at 4™C
n-Octanol (defoammg agent, BDH cat. no. 29408)
Solution B (High salt solution) 10 g Sodmm chloride (BDH, Merck Ltd. [Poole,
Dorset, UK] cat no 10241) and 1 g N-lauroylsarcosine m 100 mL of sterile deion-
ized water Adjust to pH 7.2 with 1Msodmm dihydrogen orthophosphate or 0 1N
sodium hydroxide. Store at 4°C
Solution C (For protease digestion). 1 mg Protemase K (Sigma cat no. PO390
type XI 10-20 U/mg dertved from Tritzrachzum album) m 100 mL of 0.0 IA4
Tris-HCl (BDH cat no. 27 119) Store in 1S-mL allquots at -70°C
Negative stain (for electron mtcroscopy) 2 g Phosphotungstic acid (Aldrich
[Gillmgham, Dorset, UK] cat. no 22420-o) m 100 mL of sterile deionized water
Adjust to pH 6 6 with 1ON and 1N potassium hydroxide Store at 4°C
1 Bram lysis buffer (BLB). 10 g N-lauroylsarcosme, sodium salt m 100 mL of
O.OlMsodium phosphate buffer, pH 7.4. Store at 4°C.
2 1M Tris-HCI pH 7.4: 60 57 g Tris m 450 mL distilled water Adjust pH to 7 4 with
concentrated HCl(2530 mL). Make up to 500 mL with distilled water Store at 4°C
3. 100 mA4phenylmethylsulfonyl fluoride (PMSF). 0 435 g PMSF m 25 mL propan-
l-01. Store m a dark bottle at 4°C. Care: Toxic.
4. 100 mMN-ethylmaleimide (NEM)* 0.313 g NEM m 25 mL propan-I-ol Store m
a dark bottle at 4OC Care: Toxic
5. Proteinase K (PK): 1 mg PK (Sigma cat. no P-4914) in 1 mL distilled water.
Store in 50-pL aliquots at -70°C.
6. Potassium iodide-high salt buffer (KI-HSB): 1.5 g Sodmm thiosulfate, 1 0 g
N-lauroyl-sarcosme in I mL IMTris-HCI. Add potassmm iodide IO g (for 10%)
or 15 g (for 15% solution). Make up to 100 mL with distilled water. Store at 4°C
7. 20% sucrose* 20 g sucrose m 100 mL of 10% KI HSB. REAGENTS FOR POLYACRYLAMIDE
1 Solution D: 91.5 g Trrs-HCI in 120 mL IMHCI. Adjust pH to 8.9. Make up to
250 mL wrth distilled water and filter. Store in a glass bottle at 4°C
2. Solution E: 30% Acrylamide Bis (37.5 1) (International Biotechnologres Inc ,
New Haven, CT). Make up according to manufacturer™s instructions Store m a
dark glass bottle at 4°C. Care: Toxic.
3. Solution F: 12.1 g Tris-HCl plus 4 mL 20% SDS (see thefollowing) Add 80 mL
distilled water and adjust pH to 6.8 with 1M HCl Make up to 100 mL with dis-
tilled water and filter. Store m a glass bottle at 4°C
90 Stack, Keyes, and Scott
4 Sodtum dodecylsulfate (SDS) 20 g SDS Make up to 100 mL with dtsttlled water
Store at room temperature Care: Toxic.
5 IMTrts-HCl 60 57 g Trts-HCl in 450 mL distilled water Adjust pH to 7 4 with
concentrated HCI (25-30 ml,) Make up to 500 mL with dtsttlled water Store at 4°C
6 Ammonium persulfate solution (APS) 0 14 g APS m 100 mL dtsttlled water
Make up fresh
7 Immunostammg Ku (Auroprobe BL plus Code RPN460-467, IntenSE BL Code
RPN492 Both obtamed from CAMBIO, Cambridge, UK)
8 Dtsruptton buffer 2 mL 20% SDS, 1 mL 1M Trts-HCl, pH 7 4 1 mL 2-mercap-
toethanol 0 6 g sucrose, l-2 drops bromophenol blue, 15 mL dtsttlled water
Adjust pH to 6 8 with 1M HCl Store at room temperature. Care: Toxic
9 Electrode buffer 6 g Trts, 28 8 g glycme, 10 mL 20% SDS Make up to 2 L with
distilled water
10 Blotting buffer 9 09 g Trts, 43 2 g glycme, 600 mL methanol (analar) Make up
to 3 L with dtsttlled water (pH should be approx 8.3-do not adjust )
11 12% Acrylamide separating gel (enough for one gel plate) 5 mL Solutton D,
15 8 mL Solutton E, 0 2 mL 20% SDS, 8 5 mL dtsttlled water, 10 mL APS, 11 5 pL
N,N,N™,N™,-Tetramethylethylenedtamme (TEMED). Care: Toxic
12 Stacking gel (enough for one gel plate) 1 28 mL Solution E, 1 5 mL Solutton F,
3 23 mL dtstrlled water, 6 mL APS, 3 75 uL TEMED

3. Methods
3.1. Extraction and Purification of SAF for Electron Microscopy
The extractton techniques used to detect SAF are based on provtdmg a crude tmto-
chondnal pellet that 1ssubfracttonatedto obtain synaptosomaland synaptic plasma
membrane fractions. The detergentrenders any membraneproteannonsedtmentable
at low centrifugal g-forces, but sedimentableat higher forces.The sedimentablepel-
let is treated with a proteaseenzymeand with extractionsfrom brains affected by a
TSE disease the survtvmg PrPsc can be visualized as SAF m the electron mrcroscope
The method described m this chapter conststsof extracting the CNS tissuewith
N-lauroyl sarcosmedetergent (Stage 1) and then purrfymg SAF n-rthe presence of
the enzyme ProtemaseK (Stage 2). The method is a modtficatron of two prevt-
ously published techniques (26,27). A drop of the final extract IS placed on a
formvar/carbon-coated electron mtcroscope grid, negatively stained with 2%
phosphotungtic acid and examined m the TEM mrcroscope at a magmficatton of
between 19and 30 K (Stage 3). Micrographs of fibrtls are taken on the mtcroscope,
the negativesare developed, andpnnts made,whtch aids mterpretattonof the results.
All centrtfugattons are carried out using a Beckman (Fullerton, CA) TL 100
benchtop ultracentrifuge with a 100.3 fixed angle rotor and thtck walled poly-
carbonate centrifuge tubes (max. speed of rotor 100,000 r-pm giving a g max
figure of 540,OOOg)A diagrammatic representation of each of the three stages
of the procedure is shown tn Figs. 1A-C.
Diagnosis of BSE and Scraple 91

3.1.1. Stage l-Detergent Extract/on for SAF Detect/on (Fig 1A)
1 Take 1 g of appropriate CNS tissue, place in a glass tissue grmder, and add 2 5 mL
of Solution A
2 Homogenize with at least 20 strokes of the piston and pour off mto polycarbonate
centrifuge tube
3. If the homogenate has too much foam, add two to three drops of n-octanol Leave
homogenate at room temperature for 30 min
4 Centrifuge at 22,000g average (20,000 rpm) for 10 mm
5 Gently recover the supernatant by plpeting and discard pellet
3.1 2. Stage 2-Proternase K Digestion for SA F Detection (F/g. 1B)
1 Top up the supernatant with Solution A and mix by asplratlon with a plpet
2 Centrifuge at 540,OOOg average (100,000 rpm) for 20 mm
3 Discard the supernatant and resuspend the pellet by aspiration with a plpet m 2 5 mL
of solution B
4 Centrifuge at 540,OOOg average (100,000 rpm) for 25 mm
5. Discard the supernatant and resuspend the pellet by asplratlon with a plpet m 1 5 mL
Solution C
6 Stir for 1 h at 37°C usmg a Teflon-coated magnet
7 Centrifuge at 22,000g average (10,000 rpm) for 10 mm usmg the Beckman micro-
centrifuge tubes (Eppendorf type) and the tube adaptors for the TL 100 3 rotor
8 Discard supernatant and resuspend pellet by aspiration with a plpet m 50 ML of
sterile, delomzed water.
9 MIX for 30 s using a vortex mixer

3.1 3. Stage 34egatwe Staining
for Transmission Electron Microscopy (Fig. 1C)
1 Coat 300 mesh, 3 mm diameter electron microscope grids with 0 4% formvar
(made up m chloroform) Stabilize with evaporated carbon and plasma glo treat
prior to use
2 Plpet one drop (25 pL) of the final extract onto a strip of dental wax and usmg the
Dumont EM forceps place one formvar/carbon/plasma glo-treated EM grid onto
this drop
3. Leave for 10 s, then blot, using the edge ofthe grid, on the fine grade filter paper.
4 Float the grid on 25 pL of 2% phosphotungstlc acid at pH 6.6
5 Leave for 10 s, then blot, usmg the edge of the grid, on the fine grade filter paper
Cover with a clean Petri dish and allow to dry.
6. Examine the grid m a transmlsslon electron mlcroscope at magnifications
between 19 and 30 k with an acceleratmg voltage of approx 80 Kv

3.2. Inferpretation of Results
An area of at least 20 grid squares is examined for a minimum of 20 min
before the sample IS declared negative for SAF or BSE fibrlls The morpho-
Stack, Keyes, and Scott

A 1 g I Fresh unffxed brain or spinal cord

Homogenise in 10% N-leuroylsercosine pH 7.4 I 20 strokee

------- centdfuge*
22.0009/ 10 mln -


Top up Ati 10% 3
. centMuge*
540,000 d 20 min

Resuspend in 10% NaCl
+ 1% N-lauroylsarcosine

c ;;“;:,.*
540.0009125 min -


Resuspend pellet in 1.5 ml
O.OlM TRIS + 10 ug/ml
Stir et 37OC for 1 hour
Proteinase K pH 7.2

Transfer into microcentrifuge tubes

Resuspend pellet In
Centrifuge *22,000 d 10 min
50 ul of deionised water

Fig 1. N-lauroylsarcosine and proteinase K extraction technique for the detection
of fibrils by negative stain and transmission electron microscopy. (A) Stage l-
detergent extraction. (B) Stage 2-proteinase K digestion. *Asterisk indicates
Beckman TLlOO ultracentrifuge with a TL100.3 fixed angle rotor. (continued on fol-
lowing page)
Diagnosis of BSE and Scrapie

Place formvarlcarbon coated
Pipette one drop
TEM grid (3mm diam. 300 mesh)
of final suspension
onto dental wax onto the drop and leave for 10 sets.

t V
Edge blot the TEM grid Pipette one drop of
on fine grade filter paper 2% potassium phosphotungstate (pH 6.6)
onto dental wax

Place TEM grid onto the drop Edge blot the TEM grid
and leave for 10 sets. again and allow to dry

Examine the grid in a TEM at magnifications between 20k and 40k
and at an accelerating voltage of approximately 80Kv

Fig 1. (continuedfrom previous page) N-lauroylsarcosine and proteinaseK extrac-
tion technique for the detection of fibrils by negative stain and transmissionelectron
microscopy. (C) Stage 3-negative stain procedure.

logical criteria used for the identification of SAF are those defined in the origi-
nal report of SAF in which two types of SAF were defined (2). Type I con-
sisted of two filaments measuring 24 nm in diameter, forming a fibril 12-l 6
nm in diameter with a 24 nm space between filaments. Fibrils had a marked
linearity, and were usually straight but occasionally sharply bent. They were
100-500 nm in length (although some up to 1 u were observed) and every 40-
60 nm the fibril narrowed to 4-6 nm. Type II SAF consisted of four filaments


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