a immunochemical and · stored at 4°c to await collection. hollow viscera were cut open by theatre...

Journal of Clinical Pathology, 1979, 32, 40-45

A formalin fixative for immunochemical andultrastructural studies on gastrointestinal endocrinecellsGRAHAM ROBINSON AND IAN DAWSON

From the Department ofPathology, University Hospital, Queen's Medical Centre, Nottingham, NG7 2UH,UK

SUMMARY Using indirect immunofluorescence, indirect immunoperoxidase, and unlabelledantibody enzyme techniques, gastrin, pancreatic glucagon, insulin, and somatostatin were localisedin sections of both wax- and resin-embedded tissues that had been fixed in a buffered formalinsolution. Ultrastructural preservation ofthe resin-embedded samples was also adequate for combinedelectron microscopy and light microscope immunochemistry. As the fixative concerned is stable itcan be permanently available in surgical units. It is suggested, therefore, that this fixative shouldprove useful as an alternative to buffered formaldehyde, which must be freshly prepared fromparaformaldehyde powder, in institutions where specimen collection is difficult or which have torefer cases with an endocrine involvement to other laboratories for immunochemical and finestructural examination.

The retention of the immunological activity ofantigenic determinant sites of polypeptides insatisfactorily preserved tissue is a major problemin the immunochemical demonstration of cellscontaining specific gastrointestinal hormones. Atthe light microscope level, many hormones requirespecial fixatives if reactive antigenic sites are to beretained, while others require freeze-drying andvapour-fixation with bifunctional reagents (Pearseand Polak, 1975). This latter form of tissue pre-paration gives adequate preservation for lightmicroscopy but is unsuitable for electron micro-scopy, for which buffered glutaraldehyde is preferred.This usually means either that a number of samplesmust be taken at operation and processed so thatboth light and electron microscopy can be under-taken, or that investigations are less than complete.For some hormones, this is inevitable; for othersit is not so. The antigenic determinants of severalhormones are adequately preserved in fixativesprepared from paraformaldehyde powder, andtissues so preserved are also suitable for electronmicroscopy. However, prior notice of surgery isnecessary as the fixative must be freshly prepared.Since, in many hospitals, this can give rise toproblems in obtaining suitably fresh specimens

Received for publication 26 June 1978

for fixation, this study was initiated to see if therewas available a fixative that was, firstly, stableenough to be left permanently in the operatingsuite and, secondly, suitable for both immuno-chemical and fine structural examination of tissuesamples.One possible fixative is the stable phosphate-

buffered formalin fixative introduced by Carsonet al. (1973). This fixative is reported to be suitablefor both conventional light and electron micro-scopy, and as it is stable advance knowledge ofoperation or preselection of tissue samples im-mediately after excision is not necessary. Wetherefore used it as the basis for our immunochemicalstudies on peptide hormones.

Material and methods

At operation biopsy and excised surgical specimenswere fully immersed in approximately 15 timestheir volume of Carson's fixative and were thenstored at 4°C to await collection. Hollow viscerawere cut open by theatre staff before immersionso that mucosal surfaces were exposed to fixativesolution; biopsy specimens were placed directlyinto fixative. After collection, samples of the sizerequired for subsequent light and electron micro-scope investigations were taken and re-immersed in

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A formalin fixative for immunochemical and ultrastructural studies on gastrointestinal endocrine cells

fresh precooled (4WC) Carson's fixative for furtherfixation. Total times in fixative solution variedfrom four hours for biopsy specimens to eightto 24 hours maximum for larger specimens.The fixative was prepared essentially as Carson

et al. (1973) described.

MODIFIED MILLONIG S PHOSPHATE-BUFFEREDFORMALIN (Carson et al., 1973)1 Add 186 g sodium dihydrogen orthophosphate(NaH2PO42H20) and 4-2 g sodium hydroxide(NaOH) to approximately 800 ml distilled waterand stir until the solid dissolves.2 Make up to 900 ml with distilled water and thenadd 100 ml 36% formaldehyde solution (technicalgrade).The pH of the final fixative solution is in the

range pH 7-2 to 7-4, and the osmolarity of thebuffer vehicle, that is, the salts dissolved in the dis-tilled water, approximately 290 mmol/kg.

After fixation, all tissue samples were washedfor a total of three hours in three separate changes of0-12M Millonig's phosphate buffer wash, pH 7-2,containing 3% sucrose (Robinson, 1977). Tissuefor light microscopy was then processed to 560Cmelting point paraffin wax, while samples forelectron microscopy were post-fixed for 90 minutesin Millonig's phosphate-buffered 1% osmiumtetroxide (Millonig, 1962), dehydrated in ethanol,and embedded in Araldite resin.For light microscope immunochemistry, wax

sections were cut at 5 ,um. For combined lightmicroscope immunochemistry and electron micro-scopy, the semithin-thin technique of Polak et al.(1975) was used. With this technique a 1-yimsection is cut from the resin block and mountedon a glass slide, and then several sequential ultrathinsections are cut from the same block face andpicked up on grids; the 1-,um resin section is usedfor light microscope immunochemistry and theultrathin sections for conventional transmissionelectron microscopy. Before staining, the 1-,umresin sections were etched for two minutes in asodium methoxide/benzene solution diluted 1:2with a mixture of equal parts methyl alcohol andbenzene, thoroughly rinsed in methyl alcohol/benzene (1:1) and then in absolute acetone, andfinally rehydrated to water (Mayor et al., 1961).The sequential ultrathin sections were cut at 60 nm,mounted o-l copper grids, double stained withuranyl acetate and lead citrate, and viewed in aPhillips EM 300 electron microscope.

Antisera to gastrin, insulin, pancreatic glucagon,and somatostatin (all kindly provided by ProfessorK. D. Buchanan, Department of Medicine, Queen'sUniversity ofBelfast) were fractionated to theglobulin

component using ammonium sulphate precipitation(Sternberger, 1974). After dialysis the total proteincontent of the fraction was subsequently adjustedto give 5 mg total protein/ml of dialysate. Thespecificity of the antisera was tested using immuno-electrophoresis and the double gel diffusion methodof Ouchterlony. For immunochemistry, all theantisera were diluted 1:20 with Tris-saline containing1% normal swine serum.Swine anti-rabbit IgG, peroxidase-conjugated

and fluorescein isothiocyanate (FITC)-conjugatedswine anti-rabbit IgG, soluble complex of horse-radish peroxidase (HRP) rabbit anti-horseradishperoxidase (PAP), and normal swine serum wereall obtained commercially from Mercia BrocadesLtd. For immunochemistry, the antisera werediluted with Tris-saline containing 1% normalswine serum as follows: (a) swine anti-rabbitIgG-FITC 1:16; (b) swine anti-rabbit IgG andswine anti-rabbit IgG-HRP 1:20; and (c) PAP1:60.The immunochemical staining techniques used

were indirect fluorescence, indirect immunoperoxi-dase, and the unlabelled antibody enzyme method(Sternberger, 1974; Burns, 1975; Petrusz et al.,1975).

All incubations were undertaken at room tempera-ture in moist incubation chambers, washes betweenincubations were carried out with Tris-saline(Erlandsen et al., 1974), and the peroxidase tracerwas visualised by DAB cytochemistry (Grahamand Karnovsky, 1966).

If necessary, endogenous peroxidase activitywas inhibited by pretreatment of the section withmethanol/hydrogen peroxide (Burns, 1975) ordistinguished from tracer-derived peroxidase byuse of a double staining technique (Robinson andDawson, 1975).

Control sections were incubated with each testand included specificity controls, blocking controls,and neutralising controls, which used anti-hormonesera that had been absorbed with excess antigenbefore use in the relevant staining schedule(McGuigan, 1968).

FITC-stained preparations were mounted andviewed without further treatment, wax-embeddedperoxidase preparations were counterstained withPAS/haematoxylin, and resin-embedded peroxidasepreparations were counterstained in 1% osmiumtetroxide for five minutes, washed in running water,and then stained with toluidine blue.

Results

At the light microscope level, gastrin, pancreaticglucagon, insulin, and somatostatin could all be

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Fig. 1 Glucagon-containingcells in human pancreatic islets.Wax-embedded specimen stainedby indirect immunofluorescence.x 1170

Fig. 2 Gastrin-containingcells in human pyloricantrum. Resin-embeddedspecimen stained by theunlabelled antibody enzymemethod. x 1170

localised in sections of paraffin-embedded tissuewith all the staining techniques used (Fig. 1).With etched 1-,um resin sections similar resultswere obtained, but the reaction end product ofthe PAP method was generally more intense thanthat obtained with either of the indirect techniques

(Fig. 2). Etching was an essential step in stainingprocedures with resin sections as untreated sectionswere not properly stained. Although sodiummethoxide/benzene solution used at full strengthfor one minute caused some disruption of resinsections, diluted etching solution used for two

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A formalin fixative for immunochemical and ultrastructural studies on gastrointestinal endocrine cells

minutes had no adverse effect upon the integrityof sections.

Nonspecific background staining was minimalwhen the sections had been pretreated for 10minutes with Tris-saline containing 10 % normalswine serum before application of the first immuneserum. This pretreatment blocks the cationic sites,which are responsible for nonspecific backgroundstaining (Hardy et al., 1970).There were no positively staining cells in specificity

and neutralising control sections. In blockingcontrols, some positive staining was present but theintensity of the reaction end product was minimalwhen compared with that of positive cells in testsections.

Fine structural preservation of tissues, althoughnot as good as that in glutaraldehyde-fixed tissue,was adequate for the semithin-thin technique to beused successfully (Figs. 2 and 3). The most notice-able fixation artifacts at the fine structural levelwere slight increases in width of some intercellularspaces within the gastric epithelium and a morepronounced separation between the fibrous connec-tive tissue elements in the lamina propria, especially

in the area abuttingthe mucosa (Fig. 4).

Fig. 3 Gastrin-containingcell in sequential ultrathinsection ofarea shown inFig. 2. x 5600

the basement membrane of

Discussion

In terms of tissue preservation for subsequentimmunochemistry, the endocrine cells of thegastroenteropancreatic tract can be divided intotwo groups. One group contains cells that elaboratehormones, such as GIP and VIP, whose antigenicdeterminants can be satisfactorily preserved onlyin tissue that has been snap-frozen, freeze-dried,and then vapour-fixed in a bifunctional reagent(Pearse and Polak, 1975). The second group ofcells elaborate hormones, such as the ones includedin this study, that can be localised without apparentloss of antigenicity in buffered formaldehydefreshly generated from paraformaldehyde powder.With both classes of hormones, because of thefixative methods to be used, advance knowledgeof surgery is necessary. For diagnostic and researchlaboratories which are near the operating suitethis causes no serious problems. However, whenlaboratories are some distance away from the

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Fig. 4 Gastrin-containing cell (G) with well-filled granules and a somatostatin-containing cell (D) in humanpyloric antrum. L, lamina propria. x 3920

surgical unit the collection of suitably fresh speci-mens for fixation for special procedures is oftendifficult and time-consuming, and many specimensare unavoidably air-dried before immersion infixative. This study was initiated, therefore, to seeif there was available a stable fixative for preservingthose cells normally fixed in freshly preparedbuffered formaldehyde.The results show that the buffered formalin

fixative introduced by Carson et al. (1973) meetsthe necessary criteria of being stable and suitablefor both light microscope immunochemistry andconventional transmission electron microscopy.Comparative immunochemical studies using tissuefixed in freshly prepared buffered formaldehyde andtissue fixed in Carson's buffered formalin haveshown that the fluorescent and enzyme reactionend products are of similar intensity. While at thefine structural level, although freshly preparedbuffered formaldehyde, that is, fixative less than24 hours old, gives superior ultrastructural pre-servation to Carson's formalin fixative, whenfixatives over 5 days old are used the quality of

fine structural preservation is usually better inCarson's fixed tissue than in buffered formaldehyde-fixed tissue (Robinson, unpublished observations).Although it is preferable to renew the fixative

each week, 3-month-old Carson's fixative will stillallow good immunochemical staining and produceadequate fine structural preservation (Robinson,1977).Carson's fixative should also prove useful in

those hospitals that do not have on site facilitiesfor immunochemistry and electron microscopy.Usually referred specimens have been fixed informol saline, and although immunochemistry ispossible with such tissue the results are oftenpoor and inconclusive. Fine structural preservationof such fixed wet samples is also inadequate and,when the samples provided have been embeddedin paraffin wax, ultrastructural preservation oftissue removed from the block and reprocessedfor electron microscopy is generally too poor tobe of value.

If a supply of Carson's fixative is kept in theoperating suite it will also prevent the loss of

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A formalin fixative for immunochemical and ultrastructural studies on gastrointestinal endocrine cells 45

suitable tissue which is resected at operationsundertaken out of normal laboratory hours.

Our thanks are due to those surgeons in the GeneralHospital in Nottingham, especially Mr J. Bourke,who allowed us to use material from their cases,and to Professor K. D. Buchanan for generousgifts of antisera.

This work was supported by a grant from theCancer Research Campaign, to whom we aregreatly indebted.

References

Burns, J. (1975). Background staining and sensitivity ofthe unlabelled antibody-enzyme (PAP) method.Comparison with the peroxidase labelled antibodysandwich method using formalin fixed paraffinembedded material. Histochemistry. 43, 291-294.

Carson, F. L., Martin, J. H., and Lynn, J. A. (1973).Formalin fixation for electron microscopy: a re-evaluation. American Journal of Clinical Pathology,59, 365-373.

Erlandsen, S. L., Parsons, J. A., and Taylor, T. D.(1974). Ultrastructural immunocytochemical locali-sation of lysozyme in the Paneth cells of man. Journalof Histochemistry and Cytochemistry, 22, 401-413.

Graham, R. C., Jr., and Karnovsky, M. J. (1966). Theearly stages of absorption of injected horseradishperoxidase in the proximal tubules of mouse kidney:ultrastructural cytochemistry by a new technique.Journal ofHistochemistry and Cytochemistry, 14, 291-302.

Hardy, P. H., Petrali, J. P., and Sternberger, L. A.(1970). Postembedding staining for electron micro-scopy by the unlabeled antibody enzyme method.(Abstract). Journal of Histochemistry and Cytochem-istry, 18, 678.

Mayor, H. D., Hampton, J. C., and Rosario, B. (1961).

A simple method for removing the resin from epoxy-embedded tissue. Journal of Biophysical and Bio-chemical Cytology, 9, 909-910.

McGuigan, J. E. (1968). Gastric mucosal intercellularlocalization of gastrin by immunofluorescence. Gastro-enterology, 55, 315-327.

Millonig, G. (1962). Further observations on a phosphatebuffer for osmium solutions in fixation. In 5th Inter-national Congress in Electron Microscopy, edited byS. S. Breese, Volume 2, p. 8. Academic Press, NewYork and London.

Pearse, A. G. E., and Polak, J. M. (1975). Bifunctionalreagents as vapour- and liquid-phase fixatives forimmunochemistry. Histochemical Journal, 7, 179-186.

Petrusz, P., DiMeo, P., Ordronneau, P., Weaver, C.,and Keefer, D. A. (1975). Improved immunoglobulin-enzyme bridge method for light microscopic demon-stration of hormone-containing cells of the ratadenohypophysis. Histochemistry, 46, 9-26.

Polak, J. M., Pearse, A. G. E., and Heath, C. M. (1975).Complete identification of endocrine cells in thegastrointestinal tract using semithin-thin sections toidentify motilin cells in human and animal intestine.Gut, 16, 225-229.

Robinson, G. (1977). Electron microscopy: principles ofelectron microscopy and tissue preparation. In Theoryand Practice of Histological Techniques, edited by J.D. Bancroft and A. Stevens, Chapter 20, p. 326.Churchill Livingstone, Edinburgh and London.

Robinson, G., and Dawson, I. (1975). Immunochemicalstudies on the endocrine cells of the gastrointestinaltract. I. The use and value of peroxidase-conjugatedantibody techniques for the localisation of gastrin-containing cells in the human pyloric antrum. Histo-chemical Journal, 7, 321-333.

Sternberger, L. A. (1974). Immunocytochemistry. Prentice-Hall, Inc., Englewood Cliffs.

Requests for reprints to: Dr G. Robinson, Departmentof Pathology, University Hospital, Queen's MedicalCentre, Nottingham, NG7 2UH, UK.


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