Gut, 1966, 7, 86

Histochemical study on the Paneth cell in the rat

E. 0. RIECKEN AND A. G. E. PEARSE

From the Department ofPathology, Postgraduate Medical School, London

EDITORIAL SYNOPSIS The characteristic granules of this cell fulfil the enzymatic criterion oflysosomes.

The function of the Paneth cell has more than oncebeen investigated since its discovery in 1872 bySchwalbe. A complete review of available infor-mation was presented by Patzelt (1936). Varioushistochemical approaches have been made, butknowledge about the enzymatic content of thesecells in the rat is restricted to reports on someenzymes in connexion with other investigations ofthe small intestine (Lojda and Fabry, 1959;Jervis, 1963; Liu and Baker, 1963). Studies on theheavy metal content of the Paneth cell in rats arefrequent and have been carried out more recently byMidorikawa and Eder (1962). Investigations on themembrane of the Paneth cell granules have beenperformed by Taylor and Flaa (1964). The presentinvestigation confirms and extends previous work,especially in respect of their enzyme content.

MATERIAL

Male, adult, M.R.C. hooded rats, kept on a standardlaboratory diet and tap water, were starved for five hoursand decapitated after stunning by a blow on the head.The time of sacrifice was strictly maintained constant.Various segments of the small intestine, 1 cm. in length,were quickly removed and processed in four differentways:-

1 Tissue was placed on a block-holder and quenchedimmediately in liquid nitrogen.

2 Tissue was fixed in cold 4% formal calcium for16 hours and frozen in the same way.

3 Tissue was freeze-dried in a thermoelectric freeze-dryer (Pearse, 1963) and embedded in paraffin wax afterformal-vapour fixation.4 Tissue was fixed in 70% alcohol which was saturated

with hydrogen sulphide gas, dehydrated, and embeddedin paraffin. Sections, 8 t thick, were cut on a cold micro-tome from the unfixed frozen and formalin-fixed frozenblocks; the paraffin-embedded material was cut on anM.S.E. sledge microtome (6, thick sections).

METHODS

PROTEIN Amino groups were demonstrated by the

DNFB reaction (Tranzer and Pearse, 1964) and by theAlloxan-Schiff reaction (Yasuma and Ichikawa, 1953);sulphydryl groups by the DDD-coupling method (Barr-nett and Seligman, 1952); arginine was shown by amodification of the Sakaguchi reaction (Liebman, 1951);tyrosine by the Millon reaction (Baker, 1956) and by thediazotisation-coupling method (Glenner and Lillie, 1959);and tryptophan by the method of Adams (1957). Thesereactions were carried out on freeze-dried formaldehyde-vapour-fixed material.

LIPID Neutral fat was demonstrated with oil red 0 inisopropyl-alcohol (Lillie and Ashburn, 1943) and withSudan black B in ethanol (Lison, 1953). Phospholipidswere shown by the acid haematin reaction (Baker, 1946).In addition the Kluver and Barrera (1953) method wascarried out using Luxol fast black L, Luxol fast blueARN, and Luxol fast blue G as dyes. The sections usedwere formal-calcium fixed.

CARBOHYDRATE The methods included the P.A.S. tech-nique (McManus, 1946,1948) in combination with maltasedigestion, the demonstration of acid mucopolysaccha-rides by the Alcian blue method (Steedman, 1950), theAstra blau method (Gedigk, 1956), and the reaction forsulphated mucins according to Bracco and Curti (1953).The tissues used were fixed in formal calcium.

HEAVY METALS These were demonstrated by the dithi-zone reaction according to the modification of Midori-kawa and Eder (1962) and by the sulphide-silver method(Timm, 1958), using respectively alcohol and hydrogen-sulphide gas-saturated, alcohol-fixed material.

ENZYME TECHNIQUES The following enzymes weredemonstrated:-Lysosomal enzymes Unspecific acid phosphatase

(Barka and Anderson, 1962), using ot-naphthol-ASBI-phosphate assubstrate, and, in addition, theGomori (1952)method using sodium- 8-glycerophosphate as substrate;fl-glucuronidase (Pugh and Walker, 1961) using naphthol-ASLC-fi-D-glucuronide as substrate; E 600-resistantesterase (Hess and Pearse, 1958) by the indoxylacetate-esterase technique (Holt, 1958) and by the thiolaceticacid technique described by Barrnett (1962) (inhibitor-concentration 10-3M); ,B-glucosaminidase according to

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Pugh and Walker (1960) using a-naphthyl-N-acetyl-fl-D-glucosaminide as substrate. The sections used were coldformal-calcium fixed.

Golgi-lamelle marker Thiamine-pyrophosphatase(Novikoff and Goldfischer, 1961); formal-calcium fixedsections.Microsomal enzymes Glucose-6-phosphatase (Wach-

stein and Meisel, 1956) and unspecific esterase (Holt,1958; and Barrnett, 1962), as quoted above. Unfixedcryostat sections were used.

Membrane-associated enzymes Unspecific alkalinephosphatase (Burstone, 1958) with substrate, x-naphthol-ASBI-phosphate; ATP-ase (Wachstein and Meisel, 1957)at pH7-2; leucine-aminopeptidase (Nachlas, Crawford,and Seligman, 1957) with substrate, L-leucyl-4-methoxy-,-naphthylamide. The sections were pre-fixed in thecase of the first two enzymes and unfixed in the last.

Mitochondrion-associated enzymes and coenzymesSuccinate dehydrogenase (Nachlas, Tsou, de Souza,Chang, and Seligman, 1957); NADH2 and NADPH2-diaphorase according to the method of Scarpelli, Hess,and Pearse (1958); monoamine oxidase(Glenner, Burtner,and Brown, 1957), using tryptamine hydrochloride assubstrate; Tetranitro BT was used as an electron acceptorin all the methods in this section.

Ubiquinones (Tranzer and Pearse, 1963); MTT servedas electron acceptor. Cytochrome oxidase (Nachlas,Crawford, Goldstein, and Seligman, 1958); couplingagent: 4-amino-N, N-dimethyl-naphthylamine.

Unfixed cryostat-cut sections were used.

RESULTS AND DISCUSSION

The results obtained are presented in Tables I to V.Our findings on the membrane of the Paneth granulesare essentially in accordance with those of Taylorand Flaa (1964), suggesting a carbohydrate-proteincompound as the main structural element. Inaddition, there is evidence that phospholipidsparticipate in histochemically detectable amounts.The carbohydrate compound was found to be a

neutral mucopolysaccharide which was resistant tomaltase digestion, thus excluding glycogen. Partici-pation of acidic mucopolysaccharides was ruled outby the Alcian blue and Astra blau reactions; these,and also the techniques for sulphated mucins, gavenegative results. The findings of an acidic muco-polysaccharide 'halo' enclosing the Paneth granulesin mice (Selzman and Liebelt, 1962) suggest speciesdifferences in closely related animals.The carbohydrate content is difficult to estimate

quantitatively by histochemical means. Comparisonof the P.A.S. stain in the goblet cells with the colourof the Paneth cells indicates a weak reaction in thelatter not only in formaldehyde-fixed tissues butalso in freeze-dried material. Further conclusionsas to the amount of the carbohydrate material inrelation to its classification as muco- or glycoproteincannot be made.

TABLE IRESULTS IN CARBOHYDRATE COMPOUNDS

Reaction Result

P.A.S. +P.A.S. reaction with diastase digestion +Alcian blueAstra blauReaction for sulphated mucins

TABLE IIRESULTS IN PROTEIN GROUPS

Method Result

DNFB-method for -NH, groups +Alloxan-Schiff method for -NH2 groups +DDD method for -SH groups +Sakaguchi reaction for arginine +Millon reaction for tyrosine +Diazotization-coupling method for tyrosine +Adams' reaction for tryptophan +

TABLE IIIRESULTS IN LIPIDS

Stain Result

Oil red 0Sudan Black BBaker's acid haematin method for phospholipids (+)Copper phthalocyanin +

TABLE IVRESULTS IN HEAVY METALS

Method Result

Dithizone reaction + +Sulphide silver + +

TABLE VRESULTS IN ENZYMES

Enzyme Result

Lysosomal enzymesUnspecific acid phosphatase6-glucuronidaseE 600 resistant esterase,B-glucosaminidase

Golgi apparatus markerThiamine pyrophosphatase

Microsomal enzymesUnspecific esteraseGlucose-6-phosphatase

Membrane associated enzymesUnspecific alkaline phosphataseATP-ase (pH 7-2)Amino-peptidaseMitochondrion-associated enzymesSuccinate dehydrogenaseCytochrome-C-oxidaseNADH,-diaphoraseMonoamine oxidaseUbiquinones

++(+)+++

(+)

(+?(+?

(+)(+)(+)++-1 -I-

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Analysis of the protein fraction revealed reactive-NH2 and -SH groups, tyrosine, arginine, andtryptophan. All of these exhibited slightly strongerreactivity in the Paneth granules than in the cyto-plasm of the surrounding epithelium cells. Con-sidering the number of reactive groups one mustbear in mind the various factors influencing thereactivity of proteins, especially fixation, and thepossibility of loss of reactive amino-acids duringfixation. This loss can be prevented by using freeze-dried, formaldehyde-vapour-fixed material. Histo-chemical preparations, therefore, provide a reducedpattern of active groups in the processed section.There was no evidence in our findings of the pre-ponderance of any particular reactive group asreported for arginine by Taylor and Flaa (1964).The lipid content of the Paneth cells indicated by

the techniques used was very small. Baker's acidhaematin method was positive for phospholipidsonly in some of the apical granules. A strongerreaction was obtained with the copper phthalo-cyanine method, the value of which as a histo-chemical reaction is lessened by interference fromother compounds and the fact that the colourreaction is greatly influenced by differentiation.Results similar to those using Baker's method wereobtained when Luxol fast black L was used. Noneutral fat could be shown. Taylor and Flaa (1964)also failed to demonstrate neutral fats, but Liu andBaker (1963) found in their investigations a positivestain. These controversial findings may be related todifferent functional status during the investigations.

Application of the dithizone reaction and thesulphide silver technique gave positive and com-parable results, as has been shown previously byEder (1963). The reaction product was confined tothe Paneth granules in the apical part of the cellbeing more intense at the tip than just above thenucleus (Figures 1-4). Its identification as zinc hasbeen proved spectrophotometrically after the intra-vital dithizone reaction (Stampfl, 1959), but thesignificance of this finding is not known. Activity ofa number of enzymes, such as carbonic anhydrase,some phosphatases, and others, is dependent on thepresence of zinc. In view of the highly developedenzymatic equipment of the Paneth cell granulesthis functional role of zinc is attractive, but thepossibility of elimination of heavy metals by thePaneth cell must also be considered. This is supportedby autoradiographic studies in zinc-deficient rats(Millar, Vincent, and Mawson, 1961).The investigations of the enzymatic pattern of the

Paneth cells showed a striking preponderance of thelysosomal enzymes, acid phosphatase and E 600-resistant esterase (Figures 5 and 6). On the otherhand, 3-glucuronidase was positive only when using

FIG. 1. Paneth cells of the rat jejunum; positive dithizonereaction with a maximum in the apical part of the cells.Haematoxylin and eosin. x 1,200.

FIG. 2. The same reaction as in Figure 1. x 1,200.

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FIG. 3. Paneth cells of the rat jejunum; positive sulphide-silver method showing a similar distribution pattern asFigure 1. x 1,200.

FIG. 5. Paneth cells of the rat jejunum; unspecific acidphosphatase. The maximum activity is localized in thesupranuclear zone. x 1,000.

.. 8~~~~~~~~~~~~~~~~~~~~..t.-.0

FIG. 4. The same reaction as in Fig. 3; compare also withFigure 2. x 1,200.

FIG. 6. Paneth cells of the rat jejunum; E 600-resistantesterase. x 220.

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long incubation times (two-three hours at 37°C.)and even then did not reveal much activity, generallyrestricted to a few granules.

Localization of these enzymes was clearly confinedto the Paneth granules, the distribution patternbeing the reverse of that of zinc. The strongestactivity was found in the Golgi area above thenucleus and the lowest at the tip of the cell, wherea number of granules were unstained.An attempt to demonstrate ,B-glucosaminidase

with naphthol-ASLC-glucosaminide and hexa-zonium pararosaniline failed, apparently due to thelower sensitivity of this technique. The method ofPugh and Walker (1960), using oc-naphthyl-3-glucosaminide, gave a positive reaction, however,but the localization was histological rather thancytological (Figure 7).As stated by de Duve (1963), the most important

morphological criterion for a lysosome, in con-formity with the biochemical concepts, is the demon-stration of several known lysosomal enzymes in thesame site. The Paneth cells fulfil this criterion bycontaining unspecific acid phosphatase, E 600.resistant esterase, ,3-glucuronidase, and /-gluco-saminidase. In addition acid phosphatase has beenshown ultramicroscopically by Ogawa, Masutani,and Shinonaga (1962) to be associated with thePaneth granules.Thiamine pyrophosphatase, an enzyme associated

with the Golgi apparatus, was not present at all oronly in small amounts, in striking contrast to itshigh activity in the mucous epithelium in the rat.The significance of thiamine pyrophosphatase in theGolgi apparatus is not known.

Interpretation of the significance of the E 600-sensitive microsomal unspecific esterase and glucose-6-phosphatase is difficult for technical reasons.Unspecific esterase was negative after a shortincubation time, but became positive when theincubation period was extended. This was probablydue to the appearance of E 600-resistant esteraseactivity, as could be shown by inhibition studies. Itmust be concluded that unspecific esterase waseither absent or very weak in activity. Glucose-6-phosphatase was present in the apical part of thePaneth cell (Fig. 8) but the specificity of the reactionis questionable, due to possible interference by acidphosphatase since OO1M NaF inhibited bothenzymes. Though non-specific splitting seems to beprobable it should be mentioned that in humanjejunal biopsies glucose-6-phosphatase was absentfrom the Paneth cells although acid phosphatasewas present.The membrane-bound enzymes so far tested

have given negative results. This is in strikingcontrast to the absorptive epithelium of the intestine

FIG. 7. Paneth cells of the rat jejunum; ,B-glucos-aminidase. x 450.

FIG. 8. Paneth cells of the rat jejunum; glucose-6-phosphatase. x 1,100.

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FIG. 9. Paneth cells of the rat jejunum; NADH2 dia-phorase. The diformazen deposits are mainly localized inthe basal and lateral part of the cells. A diffuse overallactivity in the apical part is due to the monoformazan.x 1,200.

which is intensely stained. Though the function ofthis enzymatic activity is still unknown it is con-

sidered to reflect active transport. Lack of theseenzymes in the Paneth cells suggests that theirprimary function is to produce a secretory product.

Recent investigations by Sylven and Bois-Svensson(1964) suggest that leucine aminopeptidase is a

lysosomal enzyme. The activity in the brush borderof the small intestine and its absence from the Panethcells made it more reasonable to associate it herewith membrane enzymes. This localization may

suggest that leucine aminopeptidase occurs indifferent cell organelles.

Oxido-reductive enzymes are predominantlylocalized in mitochondria, as shown by biochemicalinvestigations. Difficulties in histochemical tech-niques, mainly associated with the properties ofthe electron acceptors at present available, un-

fortunately set limitations to their interpretation.SDH and cytochrome oxidase, known to be mito-chondrial enzymes exclusively, showed the samelocalization as the diaphorases. The diformazan ofTetranitro BT and the crystalline reaction productof the cytochrome oxidase technique were mainlylocalized in the basal and lateral part of the cell(Fig. 9); but there was a pink colour reaction allover the cell, often staining the Paneth granulesquite intensely. This is interpreted as due to lipidsolubility of the reaction products.The heavy diformazan deposits sometimes

observed in the Golgi zone of the mucous epithelium,

after the use of Nitro BT as electron acceptor, wereabsent from the Paneth cell. Absence of this artefactcan be related to the lack of histochemically demon-strable fat in the Golgi apparatus of the Paneth cellin our study and also to the better qualities ofTetranitro BT. An occasional strong deposit ofdiformazan in the area of the Paneth granules isconsidered to be an artefact due to lipid inter-ference. The weak activity of the enzymes discussedcould reflect a low oxidation rate. Consistent withthe ultrastructural findings in mouse Paneth cells(Hally, 1958), it is more likely to be due to therelatively small number of mitochondria comparedwith the mucous epithelium of the small intestine.Monoamine oxidase localization also suffered

from lipid interference but showed a differentdistribution pattern which was on the whole similarto that of the ubiquinones. The enzyme and thecoenzyme (ubiquinone) were quite active and mainlylocalized in the apical part of the cell, as were thelysosomal enzymes (Figures 10 and 11). Theirexclusively mitochondrial localization has not yetbeen established although histochemically it is therule. Extramitochondrial ubiquinone localizationhas been noted in two other secretory cell types,i.e., salivary gland and exocrine pancreas (Tranzerand Pearse, 1964). This finding may be related tosecretory function in these cell types.

Association with the lysosomal granules cannotbe excluded but investigations on the fine structurallocalization have to be established before conclusionscan be drawn.

Interpretation of our findings with the functionalconcept of lysosomes can be readily made. As hasbeen pointed out by de Duve (1963), the functionalcycle in some cases of intracellular digestion mayacquire the character of secretion by extrusion oflysosomes, turning it into extracellular digestion.The digestive action of osteoclasts has been quotedin this connexion. The discharge of enzymaticactivity into the intestinal lumen by the Paneth cellwould make it a digestive 'gland' which participatesin the breakdown of intestinal contents. The demon-strated lysosomal activity and the presence of otherhydrolytic enzymes, not histochemically demon-strable but presumed to exist on biochemicalgrounds, would give the Paneth cell a digestivefunction of high efficiency, varying necessarily withthe number present. It is interesting to note thatthere are great differences between the variousspecies, and it seems possible that the number ofPaneth cells and their enzymatic composition isrelated to the functional demands in the animal.The extremely high quantity of Paneth cells in theTamandua tetradactyla, the ant bear, may be mention-ed here (Marques de Castro, Sasso, and Saad, 1959).

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F:

oxidase The maximum activity is localized in the apicalpart of the cell. x 1 100

1............~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

FIG. 11. Paneth cells of the rat jejunum; positive ubi-quinone reaction, localized in the apical part of the cells,mainly indicating an extramitochondrial localization.x 260.

The distribution pattern of the hydrolytic enzymesshows a typical arrangement which demands someconsideration. The highest activity is found in thegranules near the Golgi zone, the lowest at the tipof the cell. The assumption that the Golgi apparatusis the site of the production of lysosomes, wellsupported by various observations (Hally, 1958;Novikoff, Goldfischer, Essner, and Iaciofano, 1961;Becker and Sandbank, 1964), could be related to thisdistribution. Acid phosphatase activity in the Golgiarea of the human jejunal mucous epithelium occursin idiopathic steatorrhoea during treatment, indi-cating an enhanced replacement of the reducedenzyme, normally arranged beneath the brushborder (Riecken and Pearse, 1965).The high activity demonstrated in the Golgi

apparatus of the Paneth cell similarly seems toreflect a high production rate, related here to thespecial function of this cell.The distribution of zinc, on the other hand, shows

a maximum in the granules of the tip and a minimumin the Golgi area.

Relations betwen lysosomes and heavy metalshave been discussed by Daems (1962). His investi-gations suggest that iron entering the liver cells isconverted within the lysosomes into a form capableof combining with apoferritin.The reversed distribution patterns of zinc and of

the hydrolytic enzymes suggest the uptake of thezinc by the lysosomes on their way towards thetip rather than at the Golgi zone. In addition to thehydrolytic function of the Paneth cell the possiblerole of the monoamine oxidase in detoxification ofamines must be borne in mind.

SUMMARY

The Paneth cell of the rat has been investigatedhistochemically. The characteristic granules of thiscell fulfil the enzymatic criteria of lysosomes asdefined by de Duve (1963). In addition they showcertain features unusual with other lysosomes,namely, histochemical demonstrability of a carbo-hydrate-lipoprotein substance constituting the lyso-somal membrane; association with monoamineoxidase and ubiquinones and the presence of zinc.The possible role of the Paneth cell as an 'intestinal

gland' is discussed.

REFERENCES

Adams, C. W. M. (1957). A p-dimethylaminobenzaldehyde-nitritemethod for the histochemical demonstration of tryptophaneand related compounds. J. clin. Path., 10, 56-62.

Baker, J. R. (1946). The histochemical recognition of lipine. Quart.J. micr. Sci., 87, 441-470.

(1956). The histochemical recognition of phenols, especiallytyrosine. Ibid., 97, 161-164.

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Histochemical study on the Paneth cell in the rat 93

Barka, T., and Anderson, P. J. (1962). Histochemical methods foracid phosphatase using hexazonium pararosanilin as coupler.J. Histochem. Cytochem., 10, 741-753.

Barrnett, R. J. (1962). The fine structural localization of acetylcho-linesterase at the myoneural junction. J. Cell Biol., 12, 247-262.and Seligman, A. M. (1952). Histochemical demonstration ofprotein-bound sulfhydryl groups. Science, 116, 323-327.

Becker, N., and Sandbank, U. (1964). The neuronal Golgi apparatusand lysosomes in neonatal rats. J. Histochem. Cytochem.,12, 483-485.

Bracco, M., and Curti, P. C. (1953). Una nuova reazione istochimicaper l'evidenziazione delle solfomucine. Biol. lat. (Milano),6, 412-416.

Burstone, M. S. (1958). Histochemical comparison of naphthol AS-phosphates for the demonstration of phosphatases. J. nat.Cancer Inst., 20, 601-616.

Daems, W. T. (1962). Mouse Liver Lysosomes and Storage. A Morpho-logical and Histochemical Study. Drukkerijh 'Luctor et Emergo',Leyden.

de Duve, C. (1963). The lysosome concept. In Lysosomes: A CibaFoundation Symposium, edited by A. V. S. de Reuch andM. P. Cameron, pp.1-35. Churchill, London.

Eder, M. (1963). Probleme des Zinknachweises am Darm. Actahistochem. (Jena), Supplementband 3, 159-164.

Gedigk, P. (1956). Ober neue Farbeverfahren fur Mukopolysaccharideund Phospholipide. Zbl. allg. Path. path. Anat., 95, 486.

Glenner, G. G., Burtner, H. J., and Brown, G. W. Jr. (1957). Thehistochemical demonstration of monoamine oxidase activityby tetrazolium salts. J. Histochem. Cytochem., 5, 591-600.and Lillie, R. D. (1959). Observations on the diazotization-coupling reaction for the histochemical demonstration oftyrosine: metal chelation and formazan variants. Ibid., 7,416-422.

Gomori, G. (1952). Microscopic Histochemistry; Principles andPractice, pp. 189-194. University of Chicago Press, Chicago.

Hally, A. D. (1958). The fine structure of the Paneth cell. J. Anat.(Lond.), 92, 268-277.

Hess, R., and Pearse, A. G. E. (1958). The histochemistry of indoxyl-esterase of rat kidney with special reference to its cathepsin-like activity. Brit. J. exp. Path., 39, 292-299.

Holt, S. J. (1958). Indigogenic staining methods for esterases. InGeneral Cytochemical Methods, edited by J. F. Danielli,pp. 375-398. Academic Press, New York.

Jervis, H. R. (1963). Enzymes in the mucosa of the small intestine ofthe rat, the guinea pig, and the rabbit. J. Histochem. Cytochem.,11, 692-699.

Kluver, H., and Barrera, E. (1953). A method for the combinedstaining of cells and fibers in the nervous system. J. Neuropath.exp. Neurol., 12, 400-403.

Liebman, E. (1951). Permanent preparations with the Thomas argi-nine histochemical test. Stain Technol., 26, 261-263.

Lillie, R. D., and Ashburn, L. L. (1943). Supersaturated solutions offat stains in dilute isopropanol for demonstration of acutefatty degenerations not shown by Herxheimer technic. Arch.Path., 36, 432-435.

Lison, L. (1960). Histochimie et Cytochimie Animales. Principes etMethodes, 3rd ed. pp. 474-487, Gauthier-Villars, Paris.

Liu, H. Y., and Baker, B. L. (1963). The influence of technique andhormones on histochemical demonstration of enzymes inintestinal epithelium. J. Histochem. Cytochem., 11, 349-364.

Lojda, Z., and Fabry, P. (1959). Histochemie und Histologie desDunndarms der an intermittierendes Hungern adaptiertenRatten. Acta histochem. (Jena), 8, 289-300.

Marques de Castro, N. M., Silva Sasso, W. da, and Saad, F. A.(1959). Preliminary observations of the Paneth cells of theTamandua tetradactyla Lin. Acta anat. (Basel), 38, 345-352.

McManus, J. F. A. (1946). Histological demonstration of mucin afterperiodic acid. Nature (Lond.), 158, 202.

(1948). Histological and histochemical uses of periodic acid.Stain Technol., 23, 99-108.

Midorikawa, 0., and Eder, M. (1962). Vergleichende histochemischeUntersuchungen uber Zink im Darm. Histochemie, 2, 444-472.

Millar, M. J., Vincent, N. R., and Mawson, C. A. (1961). An auto-radiographic study of the distribution of zinc-65 in rat tissues.J. Histochem. Cytochem., 9, 111-116.

Nachlas, M. M., Crawford, D. T. Goldstein, T. P. and Seligman,A. M. (1958). The histochemical demonstration of cytochromeoxidase with a new reagent for the NADI reaction. Ibid., 6,445-456.-, and Seligman, A. M. (1957). The histochemical demon-stration of leucine aminopeptidase. Ibid., 5, 264-278.Tsou, K. C., Souza, E. de, Chang, C. S., and Seligman, A. M.(1957). Cytochemical demonstration of succinic dehydro-genase by the use of a new p-nitrophenyl substituted ditetra-zole. Ibid., 5, 420-436.

Novikoff, A. B., and Goldfischer, S. (1961). Nucleosidediphosphataseactivity in the Golgi apparatus and its usefulness for cytologicalstudies. Proc. nat. Acad. Sci. (Wash.), 47, 802-810.- , Essner, E., and laciofano, P. (1961). The relations betweenacid phosphatase-rich lysosomes and the Golgi apparatus.J. Histochem. Cytochem., 9, 630.

Ogawa, K., Masutani, K., and Shinonaga, Y. (1962). Electron histo-chemical demonstration of acid phosphatase in the normal ratjejunum. Ibid., 10, 228-229.

Patzelt, V. (1936). Die Oxyphilen Panethschen Kornchenzellen. InHandbuch der mikroskopischen Anatomie des Menschen, editedby W. von Mollendorff, vol. 5, pt. iii, pp. 120-137. Springer,Berlin.

Pearse, A. G. E. (1963). Rapid freeze-drying of biological tissues witha thermoelectric unit. J. sci. Instrum., 40, 176-177.

Pugh, D. and Walker, P. G. (1960). Localization of 0-N-acetyl-glucosaminidase in tissues. In Proc. 4th int. Congr. Biochem.,Vienna, 1958, vol. 17, p. 45.

- (1961). Histochemical localization of 3-glucuronidase andN-acetyl-,8-glucosaminidase. J. Histochem. Cytochem., 9,105-106.

Riecken, E. 0., and Pearse, A. G. E. (1965). Demonstration of acidphosphatase activity in the Golgi apparatus of the jejunalepithelium cell in patients with idiopathic steatorrhea. Histo-chemie, 5, 182-184.

Scarpelli, D. G., Hess, R., and Pearse, A. G. E. (1958). The cyto-chemical localization of oxidative enzymes. I. Diphosphopyri-dine nucleotide diaphorase and triphosphopyridine nucleotidediaphorase. J. biophys. biochem. Cytol., 4, 747-752.

Schwalbe, G. (1872). Beitrage zur Kenntnis der Drusen in den Darm-wandungen, in's Besondere der Brunner' schen Drusen. Arch.mikr. Anat., 8, 92-140.

Selzman, H. M., and Liebelt, R.A. (1962). The Paneth cellgranules ofthe mouse intestine. Anat. Rec., 142, 278.

Stampfl, B. (1959). Die intravitale histochemische Darstellung desZinks durch Dithizon. Acta. histochem. (Jena), 8, 406447.

Steedman, H. F. (1950). Alcian blue 8 GS: a new stain for mucin.Quart. J. micr. Sci., 91, 447-479.

Sylven, B., and Bois-Svensson, J. (1964). Studies on the histochemical'leucine aminopeptidase' reaction. IV. Chemical and histo-chemical characterization of the intracellular and stromalLNA reactions in solid tumour transplants. Histochemie, 4,135-149.

Taylor, J. J., and Flaa, R. C. (1964). Histochemical analysis of Panethcell granules in rats. Arch. Path., 77, 278-285.

Timm, F. (1958). Zur Histochemie der Schwermetalle. Das Sulfid-Silberverfahren. Dtsch. Z. ges. gerichtl. Med., 46, 706-711.

Tranzer, J. P., and Pearse, A. G. E. (1963). Cytochemicaldemonstration of ubiquinones in animal tissues. Nature(Lond.), 199, 1063-1066.

(1964). Titanous chloride as a reducing agent in the dinitro-fluorobenzene reaction for protein. J. Histochem. Cytochem.,12, 325-326.

Wachstein, M., and Meisel, E. (1956). On the histochemical demon-stration of glucose-6-phosphatase. Ibid., 4, 592.- (1957). Histochemistry of hepatic phosphatases at aphysiologic pH. Amer. J. clin. Path., 27, 13-23.

Yasuma, A., and Ichikawa, T. (1953). Ninhydrin-Schiff and alloxan-Schiff staining: a new histochemical staining method forprotein. J. Lab. clin. Med., 41, 296-299.

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