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sues at the light level and also for electron microscopy
6 For sensitive detection of gold, epi-polanzatlon of stamed se&Ions may be employed
7. One- and five-nanometers diameter particles may be enhanced with silver (lmmu-
nogold silver method) for both light and electron microscopy.
8. Adjustmg the silver mcubatlon time alters the size of the silver-gold complexes
to suit different purposes
9. Silver incubation of approx 5 min produces large silver-gold complexes that can
be seen at low magnifications allowmg the dlstributlon of the antigen to be deter-
mined over a large area of tissue (Fig 2)
10. Silver mcubatlons of approx 3 mm gives much smaller metallic aggregates that
permit more accurate locahzatlon of the antigen
11. Gold particles of lo- to 20-nm diameter can be seen readily wlthout silver
enhancement. These permit accurate locahzatlon of antigens (Fig. 3)
12 The smaller the diameter of the gold probe, the better IS the penetration of the
colloidal gold particle into the tissue.
2.1.4. Tissue Thickness
Immunostaming on routine paraffin wax-embedded tissues is generally car-
ried out on 5-7 pm thick sections. The antibodies generally penetrate the entire
thickness of the sectlon. Semlthm, plastic-embedded sections usually are only
0.5-l pm thick. This means that an antigen is only about one-fifth as abundant
in these (1 pm thick) sections when compared with routme paraffin wax-stamed
sectlons. Stamed sectlons for electron microscopy are only l/40 of the thlck-
ness of a 1-urn section (60-70 nm). Clearly, therefore, some of the patterns of
antigen dlstrlbutlon seen at light microscopy are not readily appreciated m
semlthin sections, and, m order to detect antigen in a 60-nm thick section, there
must be significant amounts of the antigen available in tissues. Tissues that
lmmunohistochemistry of Resinated Tissues 309
show only scant PrP accumulation at LM level generally are unsuttable for
studies performed at the ultrastructural level.

2.2. Specimen Protocols
2.27. Protocol for Light Microscopy lmmunostaining of PrP
in Plastic Embedded Tissues
1 FIX tissues (preferably by perfusion) in freshly prepared 3% paraformaldehydel
1% gluteraldehyde in phosphate-buffered salme (PBS), pH 7.4 (O.lM sodmm
phosphate buffer, pH 7.4) (15).
For perfused brains, remove brain and fix overnight m a fresh solutton of the
perfusate at 4°C
After fixation, dissect 1-mm cubes of brain from approprtate areas and place m
PBS, pH 7.4
Wash m PBS and postfix in 2% osmmm tetroxtde and wash m disttlled water.
Dehydrate ttssues m a graded acetone servesand mtiltrate wtth resm contamed m
beem molds.
Polymertze resm at 60°C for 48 h.
Cut sections at 1 pm and mount on poly-L-lysme coated slides or on vectabond-
treated slides (Vector Labs, Burlingame, CA)
Prepare solutton of saturated sodmm hydroxide m absolute ethanol, dtluted 1 1
wtth absolute ethanol (16) and submerge shdes in the solutton for 60 mm Satu-
rated NaOH solutton IS prepared by adding 15 g sodium hydroxtde to 100 mL
absolute ethanol, sturmg for 1 h, and allowmg the solution to age m a capped
vessel at room temperature for 5 d before decanting the supernatant for use
9. Block endogenous peroxldase for 10 min with 3% hydrogen peroxide in methanol.
10 Treat wtth 98% formtc acid for 30 mm.
11 Apply approprtate primary antibody (for example, lA8 at l/l600 dtlutton) m the
peroxtdase-anttperoxtdase lmmunocytochemical stammg procedure
12 Develop anttbody reactron product wtth 3-3™ diammobenztdme.
2.2.2. Protocol for lmmunostaining Grids
for Electron Microscopy Using an Avidin 10 nm Gold Method
1. Steps l-6 as described.
2. For serial examinations of structures identified at light mtcroscopy, take sertal 1-pm
and 80-nm sections from approprtate blocks and place latter sectrons on 400 mesh
nickel grids.
3 On grid stainmg All steps are performed at room temperature on drops on a sheet
of dental wax. Do not permtt grids to dry out throughout the entire procedure.
4. Etching step Float grids face down on a droplet of sodmm metapertodate (17)
for 60 mm (or treat with potassium methoxide 18 crown-6/Aqueous DMSO (18)
for 10 min)
5. Rinse grids m filtered distilled water for 5 x 3 mm
6. Block endogenous peroxtdase and deosmtcate wtth 3% H,O, m methanol for 10 mm
370 Jeffrey and Goodsir
7 Rinse grids m filtered distllled water for 5 x 3 mm
8 Treat with 98% formic acid for 10 mm
9 Rinse grids m filtered dIstIlled water for 5 x 3 mm
10 Rmse grids m washmg buffer before lmmunostammg for 2 x 10 mm
11 Quench residual aldehyde groups with 0.2M glycme m PBS, pH 7 4, for 3 mm,
12 Rmse the grids on washmg buffer for 5 mm
13 Incubate the grads on blockmg solution for 30 mm
14 Rinse the grids on washing buffer for 5 mm
15 Incubate grids with primary antibody (e g., a SAF lB3 diluted I 100 or 1A8 at
1.400) m mcubatlon buffer for 1 h at room temperature
16. Rinse the grids on washing buffer for 5 x 6 min
17 Incubate with blotmylated antirabbit IgG diluted I 100 m mcubatlon buffer for 1 h
18. Rmse the grids on washmg buffer for 3 x 15 mm and on PBS for 3 x 5 mm
19 Incubate with Extravldm 10 nm Colloidal Gold (Sigma, St Lotus, MO) diluted
1.10 m mcubatlon buffer for 1 h
20 Rinse the grids on washing buffer for 5 x 6 mm
2 1. Postfix grids with 2 5% gluteraldehyde m PBS for 10 mm
22 Rinse the grids on PBS for 3 x 5 mm and on excess distilled water for 2 x 5 mm
23. Counterstain with uranyl acetate and lead citrate
2.2 2 1 REAGENTS

1. Washing buffer. 0 8% bovine serum albumin (BSA) m dIstIlled water, 0 1% IGSS
quality gelatin m distilled water, 2 mM sodmm azlde (NaN,) m dIstIlled water,
adJust the pH to 7 4 with INNaOH
2. Incubation buffer 0 8% BSA m distilled water, 0 1% IGSS quality gelatm m
distilled water, 1% normal goat serum diluted m distilled water, 2 mM sodmm
azlde (NaNJ) m distilled water; adJust the pH to 7 4 with 1N NaOH
3 Blocking solution* As described, but substltute 5% normal goat serum for 1%
normal goat serum

3. Patterns of PrP Accumulation Revealed
by lmmunoelectron Microscopy
Our studies of the subcellular localization of disease-specific PrP in ME7
and 87V murme scrapie have shown that PrP accumulates inttlally at the plasma
membrane of presumed scraple-Infected cells. The antibodies employed are
not able to distmgulsh between conformationally modified PrP and normal PrP
However, we can infer that the PrP detected 1s disease specific because accu-
mulations of PrP are not found in control mice brains at the same neuro-
anatomic sites. Disease-specific PrP then spreads throughout the extracellular
matrix surroundmg scraple-infected cells and is present around many different
types of glial and neuritlc processesadjacent to the cell. Both the apparent PrP-
releasing cell and the processes around which PrP accumulates are morpho-
logically normal, at least initially With the continued accumulation of PrP,
311
lmmunohis tochemls try of Reslna ted Tissues
mdivldual amylold filaments are found within the extracellular space. At about
this time mlcroghal cell processesbecome consptcuous and possesslysosomes
that contain PrP (lO,Z9--21). Studies with N-terminal pepttde anttbodies to PrP
show that N-terminal truncation of PrP 1s not a prerequisite of amyloid fila-
ment formatton or m accumulation of preamylotd forms of PrP (Jeffrey and
Goodsir, unpublished)

References
1 Bolam, J P and Ingham, C A. (1990) Combined morphological and histochemical
techniques for the study of neuronal mrcrocircutts. Handbook Chem Neuroanat
8,125-198
2. Priestly, J. V (1984) Pre-embedding ultrastructural rmmunocytochemtstry*
rmmunoenzyme techniques Immunolab Elect Microscopy 4,37-52
3 Wiley, C A , Burrola, P G., Buchmeier, M J., Wooddell, M. K , Barry, R. A.,
Prusmer, S B., et al (1987) Immunogold localization of prion filaments m
scrapte-infected hamster brains. Lab. Invest 57, 646-656
4. McKinley, M. P , Taraboulos, A , Kenaga, L , Serban, D , Stieber, A , DeArmond,
S. J , et al. (1991) Ultrastructural locahsation of scrapte prton proteins in cyto-
plasmtc vestcles of infected cultured cells Lab Invest 65,622-630
5 Taraboulos, A , Serban, D., and Prusmer, S B. (1990) Scrapie prton proteins
accumulate m the cytoplasm of persistently infected cultured cells J Cell Bzol
110,2117-2132.
6 Caughey, B , Raymond, G. J., Ernst, D , and Race, R E. (1991) N-terminal trun-
cation of the scrapte-associated form of PrP by lysosomal protease impbca-
trons regarding the site of conversion of PrP to the protease-resistant state J Vwol
65,6597-6603
7. Caughey, B. and Raymond, G J, (1991) The scrapie-associated form of PrP 1s
made from a cell surface precursor that is both protease- and phosphobpase-sensl-
trve J BIOI Chem 266, 18,217-18,223.
8. Laszlo, L., Lowe, J , Self, T., Landon, M., McBride, P. A., Farquhar, C , et al.
(1992) Lysosomes as key organelles m the development of prion encepha-
lopathres J Pathol. 166,333-341.
9 Bruce, M E., McBrtde, P A., and Farquhar, C F (1989) Precise targeting of the
pathology of the sialoglycoprotein PrP, and vacuolar degeneration in mouse
scrapie Neuroscz Lett 102, l-6
10. Jeffrey, M., Hallrday, W. G , and Goodsir, C M. (1992) A morphometrlc and
immunohtstochemtcal study of the vestrbular complex m bovine spongrform
encephalopathy. Acta Neuropathol &I,65 1657
11. Farquhar, C F., Somervtlle, R. A., and Rttchte, L A (1989) Post-mortem
immunodtagnosis of scrapte and bovine spongiform encephalopathy. J Vzrol
Methods 24,215-222.
12 Farquhar, C F , Somerville, R A., Dorman, J , Armstrong, D., Blrkett, C., and
Hope, J. (1993) A review of the detection of PrP. Transmwtble Spongrform
312 Jeffrey and Goodsir
EncephaIopathles* Proceedmgs ofa Consultation on BSE with the Sctent$c Vet-
erinary Committee of the CEC, pp 301-303
13 Langeveld, J. P M , Farquhar, C F , Pocchlari, M., Bu-kett, C , Bostock, C., and
Meloen, R H (1993) Antlgemc sites of bovme prlon protein Transmzsslble
Sponglform Encephalopathles Proceeduzgs of a Consultatron on BSE with the
Sclentlfic Veterinary Committee of the CEC, pp 3 15-32 1
14 Kascsak, R J , Rubenstam, R , Merz, P A., Tonna-DeMasl, M , Fersko, R , Carp,
R L., et al (1987) Mouse polyclonal and moqoclonal antlbody to scraple-assocl-
ated fibrll protein J Vzrof 61, 3688-3693
1.5 Karlsson, U and Schultz, R L. (1965) Fixation of the central nervous system for
electron mlcroscopy by aldehyde perfuslon 1 Preservation with aldehyde perfu-
sates versus direct perfuslon with osmmm tetroxide with special reference to mem-
branes and the extracellular space .I Ultrstruct Res 12, 160-186.
16 Mar, H and Wlght, T N ( 1988) Colloidal gold immunostammg on deplastxrsed
ultra-thm secttons J Hlstochem Cytochem 36, 1387-l 395
17 Bendayan, M and Zollmger, M (1983) Ultrastructural locallsatlon of antlgemc
sites on osmium-fixed tissues applying the protein A-gold technique. J Hlstochem
Cytochem 31,101-109
18 Iwadare, T , Harada, E , Yoshmo, S , and Arar, T (1990) A solution for removal
of resin from epoxy sections Stazn Technol 65,205
19 Jeffrey, M , Goodslr, C M., Bruce, M E , McBride, P A , Fowler, N , and Scott,
J R (1994) Murme scraple-infected neurons m VIVO release excess PrP into the
extracellular space Neuroscr Lett 174, 39-42
20 Jeffrey, M , Goodslr, C M , Bruce, M. E., McBride, P A , Scott, J A , and
Halhday, W G (1994) Correlative light and electron microscopy studies of PrP
locahsation m 87V scrapie Bram Res 656, 329-343
21 Jeffrey, M , Goodsir, C M , Bruce, M. E , McBride, P. A., and Farquhar, C F.
(1994) Morphogenesls of amylold plaques m 87V murme scraple Neuropathol
Appl Neuroblol 20, 535-542.

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