J Clin Pathol 1982;35:1384-1387

Preparation of Epon-embedded renal tissue forscanning electron microscopyWING-LING NG, L MA, KF SO

From the Department of Pathology, University ofHong Kong, Queen Mary Hospital, Hong Kong

SUMMARY A method for the preparation of Epon-embedded renal tissue for scanning electronmicroscopy (SEM) is described. This involves careful removal of Epon by potassium hydroxide inabsolute methanol to expose the embedded kidney tissue. The method is simple and gives goodsurface morphology of the sectioned and trimmed surfaces of the tissue block. Both sectioned andintact glomeruli are well shown. The method also allows SEM observation of tubules and bloodvessels.

Scanning electron microscopy (SEM) allows thestereoscopic examination of tissues and cells and hasbeen applied to the study of human kidney tissues asearly as 1971'. Since then, many workers have lookedat both normal and abnormal kidneys under theSEM2 and several studies on the surface morphologyof the glomerulus have been reported'-8. However,there are only a few studies on the internal morph-ology of the glomerulus or tubule577. This is duepartly to the limited spatial resolution of the SEM andpartly to difficulties in tissue preparation. Severalmethods were tried to visualise the glomerularinternal surfaces including cryostat sectioning6, cryo-fracture9 and ultraplaning5. These methods,however, are unsatisfactory because they either pro-duce much tissue artefact6, require elaborate pre-paration5 or they fail to give true correlative SEMand transmission electron microscopic (TEM)observations. The difficulties can be avoided by usingtissue originally embedded in Epon for TEM". Amethod is described below in which Epon-embeddedrenal tissue previously sectioned and examined byTEM, are used for SEM.

Material and methods

Fourteen renal biopsy specimens were studied. Thesespecimens were obtained by percutaneous puncturefrom patients suspected to have glomerular diseases.The kidney tissues were processed for diagnosticpurposes for light microscopy (LM), TEM and im-

Accepted for publication 24 May 1982

munofluorescence as previously described ". Kidneytissues for electron microscopy were fixed in 2- 5%glutaraldehyde in cacodylate buffer pH 7- 4 at 4°C for1 h and postfixed in 1% osmic acid for 2 h. The tissueswere then dehydrated through graded ethanol andpropylene oxide and embedded in Epon 812. Eponblocks were processed for SEM only after routine1 ,um and ultrathin sections were made for LM andTEM for diagnosis.

REMOVAL OF EPOXY RESIN

A superficial layer of Epon, about 0 5 mm inthickness, was removed from the Epon blocks toexpose the embedded kidney tissue for SEM. Thiswas done by treating the Epon blocks with an epoxysolvent. The solvent was prepared, immediatelybefore use, by adding 5 ml propylene oxide to 10 ml10% potassium hydroxide in absolute methanol. TheEpon blocks were first hardened overnight in a 60°Coven and were then placed in glass containers, twoblocks per 5 ml epoxy solvent, which were rotatedgently for 4 to 6 min at room temperature. The blockswere examined at two-minute intervals and the Eponremoval process was stopped when about 0 5 mmthickness of the embedded kidney tissue had beenexposed. The Epon blocks were then washed in threechanges of absolute methanol and stored in absolutemethanol until required.

PREPARATION FOR SEM

The treated Epon blocks were washed in one changeof absolute ethanol followed by changes in gradedFreon TF (Dupont, USA) in absolute ethanol toabsolute Freon TF. Blocks were then air-dried at room

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Preparation of Epon-embedded renal tissue for scanning electron microscopy

temperature. The dried specimens were coated with athin layer of gold-palladium (30 nm) in a Hummer IIsputter coater (Technics, USA) and were examinedin a Cambridge Stereoscan 150 SEM (Cambridge,UK) at20kV.W,

Results and -discussion

Adequate removal of Epon appears to be essentialfor successful SEM observation of Epon-embeddedtissue originally intended for TEM. At least 0 5 mmof the renal tissue has to be exposed before good XIsurface morphology of the block can be obtained. At .low magnification by SEM, blocks with adequateremoval of Epon show not only the smooth sectioned _surface from which ultrathin sections were made butalso the slanted trimmed surfaces (Fig. 1). Renal RINglomeruli, convoluted tubules and interstitium are lreadily identified (Fig. 1). However, if only the _sectioned surface is exposed, Epon removal is likely _to be incomplete and surface morphology will beobscured by residual Epon and neither glomeruli nortubules can be recognised.

In properly prepared specimens, both sectioned Fig. 2 Sectioned glomerulus to show mesangialandintatglmerue.Sd gproliferation (M) and luminal surface ofa hilar arterioleand Intact glomeruli are seen. Sectioned glomeruli

are found mostly on the smooth sectioned surfacewhich appears clean and devoid of artefacts. The

sectioned glomeruli show intact internal architecturewith recognisable epithelial cells, capillaries andmesangial areas. Glomerular abnormalities not seenby looking at surfaces of isolated glomeruli such asinternal surface morphology of Bowman's capsule,glomerular vasculature and mesangial cell prolifera-

_YJh * > > > tion are revealed (Fig. 2). Intact glomeruli withremoved Bowman's capsules are also seen. Theseappear to have clean surfaces with minimnalextraneous deposits (Fig. 3) and changes of theepithelial cells and their foot processes can be readily

t ~~~~~~~~~studied (Fig. 4). Apart from glomerular surface*[v,_ morphology, the technique also allows SEM

observation of tubules (Fig. 5) and blood vessels (Fig.4fi6) which are well shown.

Many other preparative methods for SEM obser-vations of sectioned renal tissue have been des-cribed"7 9. None of them offers correlative SEM andTEM observations as demonstrated in our method.Furthermore these studies require separate sectionsof renal tissue and require elaborate preparation. Thepresent technique is simple and permits retrospectivecorrelate SEM and TEM studies of Epon-embedded

_u tissue.Fig. 1 Tissue blockfrom which adequate Epon has beenremoved showing smooth sectioned surface and slanted This study was supported by a grant (335/046/9965) totrimming surfaces. Convoluted tubules and glomeruli are WL Ng from the Research Grants Committee of thewell shown University of Hong Kong.

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Ng, Ma, So

iFig. 3 Intact glomerulus from Fig. I tissue block toshow wellpreserved external morphology

Fig. 5 Distal convoluted tubule to show cuboidal epithelialcells. Free surfaces ofthese cells show large numbers ofshortmicrovilli. Some exhibit a single cilium (arrow)

Fig. 4 Close-up viewofFig. 3glomerulustoshow epithelial cells with swollenfootprocesses

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Preparation of Epon-embedded renal tissue for scanning electron microscopy 1387

&&- zezS^4 Lehtonen E, Virtanen I, Wartiovaara J. Visualisation of humanglomerular changes by scanning electron microscopy. VirchowsArch [Cell Pathol] 1973;13:259-65.*1.-4 4Arakawa M, Edanaga M, Tokunaga J. Scanning electronmicroscopy of the isolated human glomerulus in normal,nephritic and nephrotic situations. In: Kluthe R, Vogt A,Batsford SR, eds. Glomerulonephritis New York: John Wiley,

5.t.B6 l SioG h-Ei ~~~~~~~~1977.-i '*..Jones DB. Correlative scanning and transmission electronmicroscopy of glomeruli. Lab Invest 1977;37:369-518.

6 Tarpey PA, Williams G. The preparation of renal tissue for: ~..~ _ G!_!- - Fl=>>'scanningelectron microscopy. Med Lab Sci 1980;37:43-56.JjEfN N d ilii ' ] t wTarpey PA, Williams G. Scanning electron microscope studies of

various glomerulopathies. Med Lab Sci 1980;37:57-80.8 Andrews PM. Scanning electron microscopy of human and Rhesus

monkey kidneys. Lab Invest 1975;32:610-8.Wheeler EE, Herdson PB. Freeze fracturing and freeze drying of

renal tissue for scanning electron microscopy. Am J Clin Pathol1973;60:229-33.

Erlandsen SL, Thomas A, Wendelschafer G. A simple techniquefor correlating SEM with TEM on biological tissue originallyembedded in epoxy resin for TEM. In: Johari 0, Corvin I, eds.Scan Electron Microsc 1973; Chicago: lIT Research Institute,1973.

Fig. 6 Close-up view ofinterstitium to show a capillary with "Ng WL, Chan CW, Yeung CK, Hua SP. The pathology of primaryintact endothelial lining. Endothelial cell nucleus (N) is seen IgA glomerulonephritis-a renal biposy study. Pathologybulging above the general surface 198113:137-43.

References

'Arakawa M. A scanning electron microscope study of the humanglomerulus. Am J Pathol 1971;64:457-66.

2 Jones DB. SEM of human and experimental renal disease. In: Requests for reprints to: Dr WL Ng, Department ofBecker RP, Johari 0, eds. Scan Electron Microsc 1979; 111. Pathology, University of Hong Kong, Queen MaryAMF O'Hare: SEM Inc, 1979. Hospital, Hong Kong.

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