Iozzo Renato V. Proteoglycan Protocols

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Cell-Mediated Transfer of PG Genes 267

type to ensure that it does not decrease proliferation and gives complete labeling as

detected by immunocytochemistry.

2. The carotid is injured as described previously but not recannulated for seeding (omit

step 20) (see Note 12).

3. A small Teflon sheet (1.5 × 3.0 cm) is placed underneath the carotid to protect surround-

ing tissue. Loose connective tissue surrounding the carotid artery should be removed.

4. The carotid artery is decellularized by gently touching liquid nitrogen-cooled forceps to the

carotid artery until frozen (several seconds). The vessel is allowed to thaw and the cycle

repeated a total of three times.

5. The Teflon sheet is removed, blood flow restored, and the decellularized carotid artery

immersed in the suspension of BrdU-labeled cells (1.25–2.5 × 10

cells/mL in 400 µL).

The cells are allowed to incubate in the peri-adventitial region of the carotid for 15 min

and then the neck incision is closed.

6. Seeded cells are identified in histological sections using anti-BrdU antibodies (Roche) and

standard immunocytochemical stains (see Note 13).

4. Notes

1. Non-species-matched genes can be used in the procedure as well. However, the develop-

ment of antibodies against the gene of interest is to be expected and might abrogate the

biological function (11).

2. In the protocols described above, we reference the LXSN vector, developed in the laboratory

of A. D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA, USA, although

procedures for transduction of related replication-defective retroviruses are similar. This vector

uses the viral long terminal repeat (L) promoter to drive constitutive expression of the gene of

interest, which is inserted into the multicloning site of the vector (X). Expression of the

neomycin phosphotransferase gene (N) is driven by an internal SV40 promoter (S) and is

responsible for the survival of the cells in the presence of the neomycin analog, G-418.

After insertion of the cDNA for the gene of interest, the orientation of the insert in the

LXSN vector should be determined by PCR or restriction digestion. Related vectors with

different promoter elements are available, including retroviral vectors that are designed to

express the gene of interest under control of a regulatable promoter.

3. PE501 and PA317 virus packaging cells (12) are grown in Dulbecco’s Minimal Essential

Medium supplemented with 10% fetal bovine serum. We suggest the sequential use of both

the mouse 3T3 cell-derived ecotropic packaging line (PE 501), which is designed for

packaging of virus that will efficiently infect mouse cells, such as the amphotropic packag-

ing line, PA317. Amphotropic virus packaging lines allow the production of virus that will

infect cells from a wide variety of different species. Well-designed packaging cell lines are

capable of synthesizing the retroviral proteins necessary for the assembly of infectious

virus, but do not release replication-competent virus.

4. Control dishes that are not infected with the virus should always be included to monitor the

selection of cells expressing neomycin phosphotransferase by G-418. In advance of

attempting viral transductions, each cell type to be used as a target must be titered against

different concentrations of G-418 to determine the concentration that will kill the cells

during the course of 10–14 d. Note that the sensitivity to G-418 varies substantially among

cells of different tissue origin or species and that, moreover, different lots of G-418 con-

tain different concentrations of active inhibitor. Note also that cells must divide in order

to incorporate the retroviral sequences into their genome. Passage of cells to relatively

low density after transduction also aids in selection, as rapidly growing cells are more

268 Fischer et al.

readily killed by the inhibition of protein synthesis by G-418. During passage, be careful

not to over-trypsinize the PA317 cells.

5. Centrifugation of the conditioned media is a precaution to avoid transfer of the virus-produc-

ing cells (PE501 or PA317) to the next cell type. Alternatively (or additionally), a 0.45-µm

filter can be used to process virus-laden medium. Media of retrovirally transduced PA317

and PE501 cell cultures contains virus particles and, although the virus is modified to be

replication-defective, all culture products must be considered Biohazardous.

6. Use the target primary cell lines at as a low passage number as is feasible. Passage 3–4

has been appropriate for endothelial cells and aortic smooth muscle cells. Typically, trans-

fection efficiency of >80% can be achieved, and the resultant pools of transduced cells

can be used without cloning. The high transduction efficiency is an advantage of this

method because it allows the establishment of transfected cell lines before cells transform

or become senescent. For experimental controls, it is always desirable to infect the target

cell line with vector alone, and to passage this control cell line in parallel with the target

cells carrying the gene of interest.

7. The procedures can be modified for the infection of larger quantities of target cells.

8. Use only freshly prepared the virus and the infection media. It is possible to repeat the

infection of target cells several times in sequence if transduction efficiency for single infec-

tions is low. Note, however, that target cells must continue to divide.

9. Cells stay viable for a couple of hours at room temperature. Ensure that cells are resus-

pended before they are instilled into the balloon-injured rat carotid artery.

10. Note that only one carotid per rat can be seeded with cells, due to vagus nerve damage,

which results in breathing difficulties for the animal.

11. The application of heparin directly before surgery can be helpful to reduce the risk of

thrombosis associated with the ballooning and cell seeding procedure.

12. Injury is not required for peri-adventitial cells to migrate through the media, but balloon

injury increases the rate of migration.

13. Cell divisions will decrease the intensity of BrdU staining.

References

1. Miller, A. D. and Rosman, G. J. (1989) Improved retroviral vectors for gene transfer and

expression. Biotechniques 7, 980–990.

2. Linial, M. L. and Miller, A. D. (1990) Retroviral RNA packaging: sequence require-

ments and implications. In Current Topic in Microbiology and Immunology. Vol. 157

Retrovirues: Strategies of Replication. (Swanstrom, R. and Vogt, P. K., eds.), Springer-

Verlag, Berlin, pp.125–152.

3. Lynch, C. M., Clowes, M. M., Osborne, W. R. A., Clowes, A. W., and Miller, D. A. (1992)

Long-term expression of human adenosine deaminase in vascular smooth muscle cells of

rats: a model for gene therapy. Proc. Natl. Acad. Sci. (USA) 89, 1138–1142.

4. Hasenstab, D., Lea, H., Hart, C. E., Lok, S., and Clowes, A. W. (2000) Tissue factor

overexpression in rat arterial neointima models thrombosis and progression of advential

atherosclerosis. Circulation, 101, 2651–2657.

5. Bonham, L., Palmer, T., and Miller, A. D. (1996) Prolonged expression of therapeutic levels

of human granulocyte colony-stimulating factor in rats following gene transfer to skeletal

muscle. Hum. Gene Ther. 7, 1423–1429.

6. Allaire, E., Forough, R., Clowes, M., Starcher, B., and Clowes, A. W. (1998) Local

overexpression of TIMP-1 prevents aortic aneurysm degeneration and rupture in a rat

model. J. Clin. Invest. 102, 1413–1420.

Cell-Mediated Transfer of PG Genes 269

7. Fischer, J. W., Kinsella, M. G., Clowes, M. M., Lara, S., Clowes, A. W., and Wight, T. N.

(2000) Local expression of bovine decorin by cell-mediated gene transfer reduces

neointima formation after balloon injury in rats. Circ. Res., 86, 676–683.

8. Clowes, M. M., Lynch, C. M., Miller, A. D., Miller, D. G., Osborne, W. R. A., and Clowes,

A. W. (1994) Long-term biological response of injured rat carotid artery seeded with smooth

muscle cells expressing retrovirally introduced human genes. J. Clin. Invest. 93, 644–651.

11. Miller, A. D., and Buttimore, C. (1986) Redesign of retrovirus packaging cell lines to

avoid recombination leading to helper virus production. Mol. Cell. Biol. 6, 2895–2902.

11. Forough, R., Hasenstab, D., Koyama, N., Lea, H., Clowes, M., and Clowes, A. W. (1996)

Generating antibodies against secreted proteins using vascular smooth muscle cells trans-

duced with replication-defective retrovirus. Biotechniques 20, 694–701.

12. Mason, D. P., Kenagy, R. D., Hasenstab, D., Bowen-Pope, D. F., Seifert, R. A., Coats, S.,

Hawkins, S. M., and Clowes, A. W. (1999) Matrix metalloproteinase-9 overexpression

enhances vascular smooth muscle cell migration and alters remodeling in the injured rat

carotid artery. Circ. Res. 85, 1179–1185.

Morphological Evaluation of Proteoglycans 271

271

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Morphological Evaluation of Proteoglycans

in Cells and Tissues

Stephanie L. Lara, Stephen P. Evanko, and Thomas N. Wight

1. Introduction

A variety of techniques are available to assess the structure of proteoglycans, their

interactions, and their locations and distribution in cells and tissues. In this chapter,

we will review the procedures that we have used over the years to examine the mor-

phology of proteoglycans and cite examples of how these techniques have been used

to address questions related to the role of these molecules in health and disease. At the

outset, we want to stress that all of the procedures we describe were developed by other

investigators, and we are merely sharing our experiences and presenting a compilation

of our favorite techniques. It should also be stressed that what follows is a small

sampling of a number of different techniques available to investigators who wish to

study proteoglycans using morphological approaches.

2. Materials

2.1. Molecular Spreads

In an effort to resolve the structural features of purified proteoglycans and the com-

plex structures that occur as these molecules interact with themselves and other mol-

ecules, we describe a spreading technique that was first used to visualize DNA (1) and

later adapted to visualizing purified proteoglycans from tissues and cells (2–4). This

method utilizes a basic protein film spread technique, which is one of several possible

approaches to preparing biochemically isolated and purified matrix molecules for

ultrastructural visualization. The approach depends on the adsorption of negatively

charged matrix molecules to a positively charged globular protein, cytochrome c,

within a hyperphase solution, and the subsequent layering of the complex onto the

surface of a hypophase. The globular protein denatures, collapses around, and coats

the negatively charged molecules and forms a strong, laterally cohesive monolayer in

which is embedded the protein-coated, negatively charged molecule. Contrast

272 Lara et al.

enhancement is achieved by staining with the heavy metals uranyl acetate and phos-

photungstate and by rotary shadowing with evaporated metal. We have used this tech-

nique to evaluate the structure of individual proteoglycan monomers as well as

proteoglycan aggregates from human intestine (5), cultured arterial smooth muscle

cells (6), bovine aorta (7), and nervous tissue (8–10).

1. Glassware: Cleaned and EtOH rinsed (see Note 1).

a. Acid-washed, thoroughly DI-H

O-rinsed glass microscope slides, stored in

90% EtOH.

b. 9-cm glass Petri dish, acid washed and thoroughly rinsed, siliconized with

Prosil-28, 1/100.

2. Samples: Buffer possibilities include 1 M NH

AC, pH 5; 0.01, 0.05 M, and 0.3 M NH

Ac,

pH 5; 0.5 M guanidine/HCl, 0.005 M benzamidine, 0.1 M 6-aminehexanoic acid, 0.01 M

EDTA, 0.0005 M phenylmethylsulfonylfloride (see Note 2).

3. Grids: Freshly prepared (2- to 3-d), very clean (acetic acid, water, EtOH rinsed), 300-mesh,

Cu grids, coated with parlodion and light carbon (see Note 3).

4. Spreading solutions

a. Hyperphase solution (prepare just before spreading): Sample diluted in appropriate

buffer, pH 5, 4°C; 1 mM Tris-HCl, 1 mM EDTA, pH 8.5; cytochrome C stock: equal

parts a and b (a. 4 M Tris-HCl, 0.1 M EDTA, pH 8.5 and b. 5 mg Cytochrome C

(purest grade)/mL H

O) (Store at 4°C up to a year.)

b. Hypophase solution may be one of the following: Deionized H

O, or 0.3 M

Ac, pH 5.0.

5. Staining solutions

a. 0.001% phosphotungstic acid in 90% EtOH, prepare fresh daily (stock: 0.1% PTA

in H

O).

b. 10 mM uranyl acetate (UA) in 90% EtOH, prepare fresh daily (stock: 5 × 10

–2

M UA

in 50 mM HCl) (Store in dark at 4°C).

6. Rotary shadowing metal: 10 mm, 8 mil gage, platinum/paladium wire.

2.2. Histochemistry of Proteoglycans—Light Microscopy

Since a majority of our work involves distinguishing regions of the extracellular

matrix (ECM) that are enriched in proteoglycans from those regions of the ECM that

contain collagen and elastic fibers, we use a modification of the Movat pentachrome

stain (11) developed by Schmidt and Wirtala (12) at the University of Washington,

(USA). This protocol involves the use of a modified Verhoeff elastic tissue stain,

which is more tolerant of minor variations in technique and does not require differen-

tiation to a subjective end point (13). This procedure is particularly useful for evaluat-

ing ECM in lung and vascular tissue (see Note 4) (14,15).

2.2.1. Modified Movat’s Pentachrome

1. Alcian blue, 1%: Alcian Blue 8GX (1 g) in distilled water (DW) (100 mL), stable for 6 mo.

2. Alkaline alcohol: 95% EtOH (180 mL), conc. ammonium hydroxide (20 mL), stable

for 6 mo.

3. Musto elastin stain

a. Alcoholic hematoxylin, 2% stock: Hematoxylin (2 g) in 95% EtOH (100 mL),

stable for 3 mo.

Morphological Evaluation of Proteoglycans 273

b. Ferric Chloride, 1.48% stock: Ferric chloride hexahydrate (2.48 m) in DW (99 mL),

conc. HCl (5 mL), stable for 6 mo.

c. Iodine stock: Potassium iodide (4 g) in DW (20 mL), iodine (2 g), DW (80 mL);

stable for 6 mo.

d. Working solution: Mix in order immediately before use 30 mL 2% hematoxylin,

20 mL 1.48% ferric chloride, and 10 mL iodine solution. Discard after use.

4. Crocein scarlet–Acid fuchsin stain

a. Crocein scarlet stock: 1 g Brilliant Crocein MOO in 99.5 mL DW, 0.5 mL conc.

acetic acid, stable for 6 mo.

b. Acid fuchsin stock: 0.1 g acid fuchsin in 99.5 mL DW, 0.5 mL conc. acetic acid,

stable for 6 mo.

c. Working solution: 4 parts crocein scarlet, 1 part acid fuchsin, stable for 3 mo.

5. Acetic acid, 1%: 1 mL glacial acetic acid, 99 mL DW, stable for 3 mo.

6. Phosphotungstic acid, 5%: 5 g Phosphotungstic acid, 100 mL DW, stable for 6 mo.

7. Saffron, alcoholic: 6 g Spanish saffron stamens, 100 mL absolute EtOH in an airtight jar.

Incubate at 60°C for 48 h to extract saffron, leave stamens in stain solution, and periodi-

cally top up alcohol over the life of the stain (1 yr).

8. Bouin’s fixative: 750 mL Saturated picric acid/H

O, 37–40% formaldehyde (Formalin)

(250 mL), 50 mL glacial acetic acid.

9. 5 µm Paraffin sections of tissue fixed in 10% neutral buffered Formalin and processed

routinely (see Note 5).

2.3. Histochemistry of Hyaluronan—Light Microscopy

This technique utilizes a highly specific peptide that interacts with hyaluronan in

tissue sections (16–18). The peptide is derived from the proteoglycan aggrecan and

can be prepared from tryptic digests of bovine cartilage by hyaluronan affinity col-

umns and then biotinylated (19). We have used this procedure to detect hyaluronan in

vascular lesions (20,21).

1. 5 µm Paraffin sections mounted on Superfrost + slides, baked 1 h at 50°C (see Note 6).

2. Xylene.

3. Graded EtOH series for rehydration: 100, 95, 70, 50, and 35% .

4. 0.7% H

in absolute methanol.

5. Phosphate buffered saline (PBS), pH 7.3: 8 g NaCl, 0.2 g KCl, 1.44 g Na

HPO

, 0.24 g

, 800 mL distilled H

O. Adjust pH to 7.3 with HCl and bring volume to 1 L with

distilled H

6. PBS/0.1% BSA: Add 1 mg of BSA (globulin-free)/mL of PBS.

7. Blocking solution: 1% BSA, globulin-free in TBS.

8. Biotinylated hyaluronan binding protein, 2–4 µg/mL PBSA (HABP).

9. Streptavidin/HRP 1:400 in PBS.

10. Tris-HCl buffer (TB), 0.05 M, pH 7.6 with 1 N HCL.

11. Diaminobenzidine (DAB).

12. Nickel chloride.

13. 30% H

14. Counterstain: 2% methyl green in NaAc buffer, pH 4.2.

15. Streptomyces hyaluronidase (1 U/µL stock).

16. Hyaluronan (for use as probe-absorbing control), add 100 µg/mL of diluted HABP and

allow to absorb overnight at 4°C.

274 Lara et al.

2.4. Intracellular Localization of Hyaluronan in Cultured Cells

We recently found that intracellular hyaluronan can be detected inside a number of

cell types (22). The cells must first be pretreated with hyaluronidase to remove peri-

cellular and cell surface hyaluronan and then permeabilized. The source of the intrac-

ellular hyaluronan, whether taken up from the pericellular matrix or synthesized within

an endosomal membrane compartment, is not yet clear.

1. Cells grown on glass coverslips in 35-mm tissue culture dishes (see Note 7).

2. 10% neutral buffered formalin.

3. PBS and PBS/1% BSA as described above for tissue sections.

4. Biotinylated HABP (100 µg/mL stock) as above.

5. Streptomyces hyaluronidase, 1 µg/µL stock solution.

6. Streptavidin–Texas red conjugate.

7. Gel-Mount fluorescence mounting medium or suitable equivalent.

2.5. Ultrastructural Localization of Hyaluronan—Scanning Electron

Microscopy

A hyaluronan-dependent pericellular matrix can be visualized around cultured

cells at the light level using the particle-exclusion method. However, the hyaluronan

network is highly soluble and fragile, and is usually lost during preparation for scan-

ning electron microscopy (SEM) when standard techniques are employed. Therefore,

we provide a simple method for preserving the hyaluronan coat for viewing by SEM to

visualize the hyaluronan filaments, which extend from the cell surface into the peri-

cellular matrix (23). Proteoglycan granules present in the cell coat can also be visual-

ized. (Also see Subheading 2.6.)

1. Cells grown on glass coverslips (as described above for intracellular localization of

hyaluronan).

2. Karnovsky’s fixative (half-strength): 2% paraformaldehyde and 2.5% EM-grade glutaral-

dehyde in 0.1 M sodium cacodylate, pH 7.3, containing 3 mM CaCl

, 5% sucrose, and

0.2% ruthenium red (RR).

3. 0.1 M Sodium cacodylate, pH 7.3, containing 3 mM CaCl

, 5% sucrose, and 0.1% RR.

4. 1% OsO

in cacodylate buffer containing 0.05% RR.

5. Streptomyces hyaluronidase (1 U/µL stock).

2.6. Histochemistry of Proteoglycans—Transmission Electron

Microscopy

A critical element in preserving proteoglycans in tissues and cells is to prevent their

solubility by including within the fixative cationic dyes that will neutralize the nega-

tive charges and make them less water soluble. One such dye that has proven to be

very effective is ruthenium red (24,25). Neutralizing the negative charges on the gly-

cosaminoglycans of the proteoglycans results in collapse of the glycosaminoglycan

chains onto the core protein and the formation of a granule that is of a size that reflects

the size of the proteoglycan monomer (4,26). An essential component of the ultra-

structural visualization of polyanionic glycosaminoglycans with cationic dyes is the

oxidation of bound ruthenium red and reduction of OsO

to an insoluble product to

provide electron density. Thus it is possible to apply the techniques of morphologic

Morphological Evaluation of Proteoglycans 275

stereology to estimate quantitatively the content of proteoglycans that are spatially

preserved in tissue sections (26–29).

1. 0.2% ruthenium red (RR) in half-strength Karnovsky’s fixative (2% paraformaldehyde

and 2.5% EM-grade glutaraldehyde in 0.1 M NaCacodylate, 3 mM CaCl

, pH 7.3) (4,26).

2. 0.1% ruthenium red in 0.1 M NaCacodylate, 3 mM CaCl

, 3% sucrose, pH 7.3.

3. 0.05% ruthenium red in 1% OsO

in 0.1 M Cacodylate, 3 mM CaCl

, pH 7.3 (see Note 8).

4. 0.1 M NaCacodylate, 3 mM CaCl

, pH 7.3.

5. Graded EtOH series: 35, 50, 70, 90, 95, and 100%.

6. 3% uranyl acetate in 70% EtOH, stored in dark, filtered before use.

7. Propylene oxide.

8. Epoxy resin: Mix thoroughly in this order: 29.0 g Eponate; 16.g DDSA; 14.3 g NMA.

Add and mix thoroughly, BDMA 1.18 g. Store frozen in air-tight 20-mL aliquots (see

Note 9).

9. 7% (saturated) uranyl acetate in dd-H

10. Reynolds lead citrate: 2.66 g Lead nitrate, 3.52 g Sodium citrate, 60 mL fresh dd-H

shake 5 min until dissolved, then invert every 5 min over the next 0.5 h. Add 1 N NaOH

(freshly made, 1 g/25 mL dd-H2O) (16 mL), bring volume to 100 mL with dd-H

2.7. Immunocytochemistry of Pr-oteoglycans—Light Microscopy

During the last 5 yr there has been an explosion of available antibodies to virtually

all of the core proteins of the known proteoglycans as well as to several glycosami-

noglycan epitopes. This has provided the opportunity to “map” the specific location

of particular proteoglycans to defined regions in tissues and associated with cells.

Defining the precise location of a proteoglycan in tissue sections aids in identifying

different functions of specific proteoglycans (21,30–33). In addition to the use of paraf-

fin processed tissue and avidin–biotin detection systems for the immunolocalization of

proteoglycans, we have found it useful to process tissue with the goal of visualizing

tissue antigens at both the light and electron microscopic level on the same section or on

adjacent sections. This can be done by processing tissue for electron microscopy and

removing the plastic resin following sectioning for light microscopy (15,34). Below

we describe two different procedures we use to localize proteoglycans in tissue sec-

tions.

2.7.1. Paraffin

1. 5 µm paraffin sections mounted on Superfrost + slides, baked 1 h, 50°C (see Note 10 and

Note 11).

2. Xylene.

3. Graded EtOH series for rehydration: 100, 95, 70, 50, and 35%.

4. 0.7% H

in absolute methanol.

5. Tris-HCl buffer (TB), 0.05 M, pH 7.6 with 1 N HCL.

6. Tris-HCl buffered saline (TBS), 0.9% NaCl, pH 7.6.

7. Phosphate–buffered saline (PBS), pH 7.3: 8 g NaCl, 0.2 g KCl, 1.44 g Na

HPO

, 0.24 g

, 800 mL distilled H

O. Adjust pH to 7.3 with HCl and bring volume to 1 L with

distilled H

8. PBS/0.1% bovine serum albumin: Add 1 mg BSA (globulin-free)/mL PBS (see Note 12).

9. Blocking solution: 10% normal serum (corresponding to secondary antibody species),

1% bovine serum albumin, globulin-free (BSA) in TBS.

276 Lara et al.

10. Diluent: 0.1% BSA in TBS.

11. Primary antibody: Diluent, dilution determined by titration on positive control tissue.

12. Biotinylated secondary antibody, 1/200 diluent.

13. Vectastain Elite ABC reagent, vector, solutions A and B.

14. Diaminobenzidine.

15. Nickel chloride.

16. 30% H

17. Counterstain: 2% methyl green in sodium acetate buffer, pH 4.2.

18. Enriched Tris buffer, pH 8.0: 0.6 g Tris base, 0.8 g sodium acetate, 0.3 g NaCl, 100 mL

distilled H

O, adjust pH with 10 N HCl, add BSA (1 mg/mL).

19. Enzymes: Chondroitin ABC lyase (0.2 unit/mL enriched Tris buffer) trypsin (1 mg/mL

0.2 M Tris, 4 mM CaCl

, pH 7.7).

2.7.2. Plastic Removal

TISSUE FIXATION AND EMBEDDING IN PLASTIC

1. 0.2 M PO

buffer: To 405 mL of 0.2 M Na

HPO

(28.39 g/L), add approx 95 mL of 0.2 M

NaH

•H

O (27.6 g/L) to give a pH of 7.4. Add distilled H

O to give final volume of

500 mL.

2. Fixative: 3% paraformaldehyde, 0.25% EM-grade glutaraldehyde in 0.05 M PO

buffer:

In a fume hood stir 3 g of paraformaldehyde into 20 mL of 65°C distilled H

O, add 2–3

drops of 1N NaOH and stir until clear, then cool. Mix 25 mL of 0.2 M phosphate buffer,

50 mL distilled H

O, 0.5 mL of 50% EM-grade glutaraldehyde, and cooled paraformalde-

hyde. Adjust pH to 7.4 and bring final volume to 100 mL with d-H

3. Phosphate–buffered saline (PBS): 0.05 M phosphate buffer, 0.9% NaCl, pH 7.3.

4. 0.1 M glycine in PBS.

5. Graded ethanol series: 35, 50, 70, 90, 95, and 100% (absolute).

6. 3% uranyl acetate in 70% EtOH, stored in the dark and filtered with #1 Whatman filter paper

before use.

7. Epoxy resin: Mix equal parts of solution A (50 mL of DDSA and 37.5 mL of EPON 812,

well mixed) and solution B (50 mL of EPON 812 and 36.7 mL of NMA, well mixed). Add

12 drops of DMP-30 for each 10 mL of resin and stir thoroughly (see Note 13).

SECTIONING, PLASTIC REMOVAL, AND IMMUNOSTAINING

1. Superfrost plus microscope slides.

2. Saturated Na ethoxide: Put 15 g of NaOH in a 100 mL plastic bottle, fill with absolute

EtOH and allow to dissolve and age 2–3 d on a shaker. The resulting solution will be a

light tea color.

3. Absolute EtOH.

4. Graded ethanol series for hydration.

5. 0.3% H

in absolute methanol.

6. Immunoreagents as in Procedure 1—Paraffin.

2.8. Immunocytochemistry—Transmission Electron Microscopy

The successful immunodetection and localization of specific molecules within

tissue prepared for ultrastructural examination is subject to a number of factors,

including epitope maintenance during fixation and tissue/reagent penetration

limitations. The physical and chemical nature of individual epitopes within the

microenvironment of the tissue or culture condition of interest determine the

Morphological Evaluation of Proteoglycans 277

impact of these factors and ultimately the choice of detection protocols. No single

processing procedure can be recommended, but preliminary light microscopy

establishes some of the limitations to be encountered and provides direction. If a

molecule cannot be detected using light microscopy, ultrastructural localization is

unlikely to be achieved. There are a number of different ways to prepare tissue and

cells for the ultrastructural examination of proteoglycan location. Below, we

describe two different methods that we have used with considerable success to

examine proteoglycan localization at the ultrastructural level. In Procedure 1 the

acrylic monomer resin LR White is used because its hydrophilic nature allows the

penetration of immunochemicals without plastic removal or etching, in contrast to

epoxy resins, avoiding possible alteration of antigenic sites with removal or

etching agents (15,35,36). In Procedure 2 the removal of plastic from sections of

epoxy resin-processed tissue has the effect of “unmasking” previously inacces-

sible epitopes and allowing a precise correlation of light and EM immunostaining

(37,38).

2.8.1. Procedure 1: LR White (LRW) Resin

TISSUE FIXATION AND EMBEDDING (

SEE

NOTE 14)

1. Phosphate buffered saline (PBS), pH 7.3: 8 g NaCl, 0.2 g KCl, 1.44 g Na

HPO

, 0.24 g

, 800 mL distilled H

O. Adjust pH to 7.3 with HCl and bring volume to 1 L with

distilled H

2. Fixative: 3% paraformaldehyde, 0.25% EM-grade glutaraldehyde in PBS, pH 7.3, freshly

prepared.

3. 0.3 M glycine in PBS.

4. 30%, 50%, and 70% methanol and 80% EtOH (see Note 15).

5. 2% uranyl acetate (UA) in 70% methanol, store in the dark (36) (see Note 16).

6. 2/1 mixture LR White/80% EtOH.

7. LR White acrylic resin (medium or hard).

8. Gelatin capsules.

9. Reverse carbon-coated formvar substrated Ni grids, 200 mesh.

IMMUNOLABELING AND POSTSTAINING

1. 80 to 100-nm LRW-embedded sections mounted on formvar-coated Ni grids (see

Note 17).

2. Tris-HCl buffered saline (TBS): 50 mM Trizma base, 0.15 M NaCl, pH 7.6 with 1 N HCl.

3. Blocking buffer:10% normal serum, 1% BSA in TBS.

4. Diluent/wash buffer: 0.1% Tween 20, 0.1% BSA, 0.1% NaN

, TBS, pH 8.2.

5. Primary antibody diluted in diluent/wash buffer and microfuged for 1 min just before use

(see Note 18).

6. 10-nm colloidal gold-conjugated secondary antibody diluted in diluent/wash buffer and

microfuged 1 min just before use.

7. 3% glutaraldehyde in PBS.

8. 7% uranyl acetate in dd-H

9. Reynolds lead citrate: 2.66 g Lead nitrate, 3.52 g Sodium citrate, 60 mL H

O; shake 5 min

until dissolved, then invert every 5 min over the next 0.5 h, add 16 mL 1 N NaOH (freshly

made, 1 g/25 mL dd-H2O), bring volume to 100 mL with dd-H

10. Aqueous 2% OsO