Deamination to prevent the competitive effect of proteins in the metachromatic staining of sections...

Histoehemie 8, 252--263 (1967)

DEAMINATION TO PREVENT THE COMPETITIVE EFFECT OF PROTEINS IN THE METACHROMATIC STAINING

OF SECTIONS AT LOW pH-VALUES

HELGE ANDERSEN and NIELS EHLERS Laboratory of Cyto- and Histochemistry, Department of Human Anatomy (Institute A),

University of Copenhagen, Universitetsparken 1, Copenhagen 0, Denmark

Received October 28, 1966

Summary. To prevent the competitive cation effect of present proteins in the utilization of the effect of pH in differential histochemical staining of mucopolysaccharides with cationic dyes, preliminary oxidative deamination with 0.5 % ninhydrin in absolute ethanol at 37 o C was carried out. Thirty minutes deamination was sufficient to prevent the competitive effect of proteins at low pH values (1.0), even in tissue (cornea) with a high content of proteins compared with the content of acid mucopolysaccharides (glycosaminoglycuronoglycans).

Introduction

The interaction of tissue polyanions with cationic dyes is of electrostatic nature and consequently, it is generally supposed that this interaction depends on the hydrogen ion concentration and on the pK value of the anion groups. This has led to the use of metachromatic staining at varying pH values in the demonstration of sulphate, phosphate, and carboxyl groups for the purpose of distinguishing between sulphated mucopolysaccharides [chondroitin sulphates and keratosulphatc (keratan sulphate)], nucleoproteins, and hyaluronic acid (SHcER, 1962).

Experiments in vitro with pure solutions of chromotropic tissue substances give fairly unequivocal results, as shown by SZlRMAI (1963) and accordingly, it would be expected that carboxyl groups were not dissociated below pH 4.0 and at least had ceased to be electronegative at pH 2.0. Similarly, phosphate groups stop being electroncgativc between pH 1.0 and 2.0, whereas sulphate groups are still electronegative and consequently are chromotropic below pH 1.0.

Conversely, experiments in vivo are complicated on account of many different factors, the main features of which were clearly formulated by KELLY (1958): "Since chromotropes form unknown complexes in biological materials which are further modified by the histologists' technical insults, it is not possible to speak of isoelectric ranges of the complexes or the dissociation constants of the anionic groups". Of these factors, dye-binding at varying pH values to non- fixed tissue, which was studied extensively by SZmMAI and BALAZS (1958), is characterized by two different, not independent phenomena - - dye-binding and metachromasia. Furthermore, these investigators discussed the stoichiomctry of the reaction. Also the presence of proteins plays an important role in the metachromatic reaction, as shown by KELLY (1955): FOLLIS (1951), HAMERMAN and SCHUBERT (1953), FRENCH and BENDITT (1953), UDUrA and DUNPHY (1956), NOGUCHI (1956), SZlRMAI (1963) and CURRAN (1964). In particular at low pH values the proteins will compete with the cationic dyes used in the metachromatie

Proteins in the Metachromatic Staining 253

reaction. Any excess of proteins m a y block the chromotropic tissue anions completely. A similar compet i t ive effect m a y be exhibi ted by salt cat ions which,

however, have l i t t le or no influence on the me tachromat i c react ion in ord inary tissue ,sections, since under normal condit ions these cations are washed out of

the section dur ing the process of preparat ion. Mineralized tissues form an excep- t ion, as e.g. bone, cart i lage, and too th mat r ix , where calcium ions inhibi t comple-

te ly the me tach romat i c react ion which can be restored only af ter decalcif icat ion (ScHAJOWICZ and CAB~I~I, 1955; CABRI~I, 1961; ANDrnS~?r 1963 ; MATTHIESS~N, 1967).

The present s tudy relates solely to the competitive e/[ect o~ tissue proteins on demonstrating acid mucopolysaccharides (glycosaminoglycuronoglycans) , and the

aim was to develop and s tandardize a me thod whereby this competitive e//ect o~ the proteins could be prevented or reduced so tha t i t would be possible to ma in ta in the same concent ra t ion of dye cations during the metachromat ic s taining a t va ry ing p H values.

Materials and Methods

Cornea and aorta from monkeys (Cereopithecus ethiops) were used as well as tissues from human foetuses (the cartilagineous head of femur, lower limbs, and umbilical cords) from the first half of the prenatal period. The tissue was removed and fixed within 30 min post mortem, or 30 rain after the foetuses were removed by legal abortion.

Fixation. Since rigorous directives cannot be established for the fixation of acid mucopolysaccharides, the following fixatives and mixture of fixatives were used: 1.4% buffered neutral formaldehyde, 2. Lillie's ethanol-formalin-acetic acid (LILLIE, 1954), and 3. cetylpyridinium chloride-formalin (WIr~IAMS and JACKSOn, 1956). As regards the first two fixatives, ice-cold reagents were used, and the fixation proper was carried out at 0--4 o C. The third fixative was applied at room temperature. The experiments showed that excellent and almost identical results were obtained by all three methods. Cetylpyridinium chloride-formalin was employed as the standard fixative, since cetylpyridinium chloride combines with acid mucopolysaccharides forming quarternary ammonium complexes which are insoluble in aqueous solutions of low ionic strength. Furthermore, it is possible to carry out comparative experiments with ordinary paraffin sections and frozen sections, since the latter can be cut immediately after fixation and be transferred directly from the microtome to a bath of distilled water of low ionic strength in order to remove any excess of fixative. Subsequently, the sections can be flattened directly and dried on slides without using adhesive agents. In both methods the sections were cut at 8 microns.

Histochemical methods: 1. Metachromatic staining with 0.1% toluidine blue (MERCK) in 30% ethanol. 2. Metachromatic staining with 0.1% toluidine blue in McIlvaine buffer (pH 2.2, 3.0,

4.0, 5.0, 6.0, and 7.0). 3. Metaehromatic staining with 0.1% toluidine blue in Walpole buffer (pH 1.0). 4. Metachromatie staining with 1% toluidine blue in Walpole buffer (pH 1.0). 5. Staining with 0.1% Aleian blue (G. T, GURR) in 3% acetic acid (pH 2.7---3.0). Staining

time: 30 rain (Mow~Y, 1956). 6. Staining with 0.1% Alcian blue in Walpole buffer (pH 1.0). Staining time: 30rain. As regards the metachromatic reactions it applied that after staining (staining time:

2 rain at pH 5, 6, 7, and in alcoholic toluidine blue solution; 10 rain at the other values of pH, 2 min, however, in 1% solution of toluidinc blue), the sections are rinsed twice in distilled water and afterwards transferred directly to two shifts of absolute ethanol (brief rinse and 3 min, respectively). Subsequently xylen and mounting in DePex.

7. Oxidative deamination with ninhydrin (0.5% ninhydrin in absolute ethanol) at 370 C for 15 rain, 30 rain, 1 hour, 2 hours, 6 hours, and 20 hours.

17"

254 H. ANDERSEN and N. EHLERS:

8. Deamination at 0--4 ~ C for 48 hours by means of a mixture of equal volumes of 10% acetic acid and 5% sodium nitrite (PEARSE, 1960).

9. Methylation (LILLIE, 1954 and 1958; FISCHER and LILLIE, 1954) in methanol-HC1 for one hour at 580 C.

10. Pepsin digestion with 2 mg/ml of pepsin crystallized three times (Nutritional Bio- chemicals Corp., Cleveland) in 0.02 n HCI at 370 C for 30--60 min (PgARSE, 1960).

11. Digestion with testicular hyMuronidase (Fluka) (PEARSS, 1960). 12. Yasuma and Itchikawa's ninhydrin-Schiff method for proteinbound NH, z (PEARSE,

1960) (KAsTEN, 1962).

Observations

The course of exper iments was as follows: I . Spot tests . I I . Combined spot and tissue exper iments . I I I . Tissue exper iments . IV. Control exper iments .

I. Spot tests

Vary ing mix tures of egg a lbumin, h u m a n y-globulins, and chondroi t in su lphate were smeared on clean slides, a i r -dr ied and f ixed in formal in vapour for 30 - -60 min. A mix tu re of 0.25% chondroi t in sulphate , 5.0% egg a lbumin, and 2.5% h u m a n y-globul in p roduced a f i lm exhib i t ing a p ronounced me tachromas ia when s ta ined with 0.1% toluidine blue in 30% ethanol (2 rain), whereas the me tach roma t i c reac t ion was negat ive a t s ta ining with 0.1% toluidine blue a t p H 1.0 (10 min). Deamina t i on with 0.5% n inhydr in in absolute e thanol for 30 min a t 370 C and subsequent r insing in t a p wate r for 2 to 3 rain pr ior to the me tachromat i c s ta ining p roduced ident ical resul ts when s ta ined with 0.1% toluidine blue in 30% ethanol , b u t fa i r ly p ronounced me tach romas i a a t s ta ining a t p H 1.0. Control exper iments wi th the n inhydr in-Schi f f me thod appl ied to the smears showed an ex t r eme ly s t rong reac t ion indica t ing the presence of large quant i t i es of pro te in -bound N H 2.

II . Combined Spot and Tissue Experiments

Some fresh h u m a n umbi l ica l cords were in jec ted with an aqueous solut ion conta in ing 0.1% of egg a lbumin and 0.1% of gelat ine, while others were in jec ted wi th a solution conta in ing 6.6 % of a lbumin and 3.3 % of h u m a n y-globulin. Af ter in jec t ion the t issues were f ixed for 24 hours in ce ty lpyr id in ium chloride-formalin. F rozen sections and o rd ina ry paraff in sections were then produced. Non- in jec ted umbi l i ca l cords were used as controls.

F rozen sect ions of in jec ted umbil ical cords showed pronounced me tachromas ia in the s t roma and vascu la r walls when s ta ined with 0.1% toluidine blue in 30 % ethanol . Metachromat ic s ta ining a t va ry ing values of p H showed a cont inuously decreasing me tachromas ia below p H 4.0, d i sappear ing comple te ly a t p H 1.0. Conversely, sections which were t r ea t ed with n inhydr in for 30 min a t 37 o C before s taining, exh ib i t ed me tachromas ia a t p H 1.0. The react ion was most p ronounced in the vascu la r walls (the arteries) and less pronounced in the gelat in- ous umbi l ica l cord s t roma and in the wall of the vein.

Alcian blue s ta ining a t p H 2.7 p roduced a similar p ronounced s ta ining of the vascu la r walls and the umbil ical cord s t roma, whereas s ta ining at p H 1.0 showed


a weaker reaction. Previous ninhydrin t rea tment for 30 min produced no changes in the reaction with Alcian blue staining at p H 2.7, whereas at pH 1.0 the reaction was enhanced considerably.

In paraffin sections the conditions were similar to those seen in frozen sections, but the ninhydrin effect was less pronounced at the low p H value (1.0) than in the corresponding frozen sections.

The ninhydrin-Schiff test for protein-bound NH~ showed a distinctly positive reaction in the stroma of the umbilical cords injected with protein, whereas control sections of noninjected umbilical cords showed negative reaction in the stroma and positive reaction was seen only in the cytoplasm of the smooth muscle cells of the vascular walls.

The control sections of non-injected umbilical cord showed constantly decreasing metachromasia below p H 4.0, but were still metachromatic at pH 1.0. Like- wise a fairly pronounced degree of staining with Alcian blue was seen both at pH 2.7 and 1.0. These conditions did not change after ninhydrin t reatment .

I I I . Tissue Experiments a) Cornea. Following toluidine blue staining at p H above 4.0, a pronounced

metachromasia of the corneal s t roma was seen both in ]rozen sections and in para]/in sections, whereas the metachromasia disappeared completely at pH below 4.0 even if the staining time was prolonged. Ninhydrin t rea tment of the sections for 30 min before staining with 0.1% toluidine blue, however, gave a pronounced metachromatic reaction at p H 1.0 (Fig. 1), just as this metachromasia could be produced at pH 1.0 in non-ninhydrin treated sections by increasing the concentration of toluidine blue to 1%.

Alcian blue staining produced a positive reaction at pH values of both 2.7 and 1.0, and this reaction was nearly unchanged by preceding ninhydrin treatment.

With the ninhydrin-Schiff test for protein-bound NH~, a positive reaction was observed in the corneal stroma.

After pepsin digestion of the corneal s troma it was possible to produce a fairly constant metachromasia by staining at p H values of 4.0, 3.0, 2.2, and 1.0.

Hyaluronidase digestion of the cornea, which had been exposed to ninhydrin t rea tment for 30 min, showed slight decrease in the metaehromatic reaction of the corneal s t roma at pH 1.0 and with 0.1% toluidine blue in 30% ethanol, which, as regards the latter, is in accordance with the findings in previous studies (E~rL~S, 1966).

b) Aorta. In/rozen sectio~ a strong metaehromatie reaction was seen in the interfibrillar substance with 0.1% toluidine blue in 30% ethanol, whereas the reaction was completely negative at pH 1.0. When the metachromatic staining at p H 1.0 was preceded by ninhydrin t rea tment for 30 min, almost the same pronounced metachromasia was produced as that seen with 0.1% toluidine blue in 30% ethanol (Fig. 2).

The para/]in sections showed the same results as the frozen sections when stained with 0.1% toluidine blue in 30% ethanol, whereas staining at p H 1.0 produced a fairly weak metachromatic reaction in the interfibrillar substance. After 30 min ninhydrin t reatment , this weak reaction had increased to almost the strength seen after staining with 0.1% toluidine blue in 30% ethanol.

256 H. ANDERSEN and N. EKLERS:

With Alcian blue the intercellular substance gave pronounced reaction at pH 2.7--3.0 and a somewhat weaker reaction at pH 1.0. The reaction at pH 1.0 was markedly increased after ninhydrin treatment for 30 min.

The ninhydrin-Schiff reaction for protein-bound NH 2 was positive, corresponding to the elastic fibres.

c) Human Foetal Lower Limb. The joint interzone displayed a constant strong metachromasia in the intercellular substance at metachromatic staining down

Fig. 1 a--e . Frozen sections of cornea after f ixat ion in cetylpyridinium chloride-formalin for 24 hours, a Staitied with 0.1% toluidine blue in 30% ethanol, b Stained with 0.1% toluidine bhm in Walpole btfffer (pH 1.0). c Stained with 0.1% totuidinc blue in Walpole buffer (pH 1,0) after 30 rain oxidat ive deamination with 0.5 %

ninhydrin in absolute ethanol

to pH 1.0, whereas the metachromatic reaction in the ordinary vascular mesenchyme around the joint region disappeared completely within the pH range of 3.0--4.0. Pretreatment with ninhydrin for 30 min did not alter these conditions, and presumably this is due to the fact that at this stage of development the intercellular substance of the joint interzone consists almost exclusively of chondroitin sulphate A or C (or possibly both), whereas the intercellular substance of the surrounding vascular mesenchyme consists mainly of hyaluronic acid (ANDER- SEN, 1964). The hyaline cartilage displayed constant metachromasia down to pH 1 and this was not changed after pretreatment with ninhydrin for 30 min. This is in accordance with the fact that at these early stages of developmentthe

Proteins in the lVIetachromatic Staining 257

cartilage consists almost exclusively of chondroitin sulphate - - connected in some way with certain slightly PAS-positive substances.

IV. Control Experiments With the object of investigating, whether the effect of ninhydrin was actually

an oxidative deamination of the present proteins and not a fortuitous induction of tissue anions, the following control experiments were carried out:

Fig. 2a---c. Frozen sections of aorta. Fixat ion in cetylpyridinium chloride -formalin for 24 hours, a, b, and c stained as :Fig. l a , b, and c, respectively

a) Foetal hyaline cartilage (head of the femur) was methylated, whereby the metaehromasia disappeared completely. Other methylated sections were treated with ninhydrin for 30 min at 37 o C before staining with 0.1% toluidine blue in 30 % ethanol and with 0.1% tolnidine blue at pH 1.0. In none of the cases did the sections display a metachromatic reaction. Furthermore, smears of pure protein solutions were treated with ninhydrin for 30 rain at 37 ~ C. Subsequent staining with tolnidine blue did not produce metaehromasia.

b) In order to investigate further the effect of the alcoholic solution of ninhydrin, control sections were incubated simultaneously for 30 min at 37 ~ C in absolute ethanol. The experiment showed ninhydrin to be the active substance.

c) In order to establish that the effect of ninhydrin was a deamination of the tissue proteins, control sections were deaminated with nitrous acid for the

258 H. AND~S~ and N. EHLERS:

purpose of comparing with the ninhydrin-treated sections. Subsequent metachromatic staining showed that both types of deamination produced identical results.

d) In order to establish the opt imum time interval for the oxidative deamination with ninhydrin ~t 370 C, experiments were carried out at varying intervals: 15 min, 30 rain, 1 hour, 2 hours, 6 hours, and 20 hours. The experiments showed tha t deamination of the tissue for 15 to 30 rain was sufficient and produced a minimum degree of other changes in the tissue. Ninhydrin t reatment for 30 rain produced the same nucleoprotein staining in the cytoplasm of the cells of the corneal epithelium by subsequent application of toluidine blue as in sections which had not been treated with ninhydrin, whereas prolonged t reatment with ninhydrin resulted in a green (bathochrom) nucleoprotein reaction at subsequent toluidine blue staining.

Discussion and Conclusion

The chemical background for the apphcation of the metachromatic reaction as a histochemical method has been the subject of extensive research (BANK and BUNGENBEllG DE JONG, 1939; HAM~MA~ and SCnVB~T, 1953; KRAMER and WI~DRUM, 1955; SC~UBEI~T and HAMERMA~, 1956; BI~GERON and SINGER, 1958; BOOlJ, 1958; SCH]~IBE and ZA~KER, 1958; KELLY, 1958; SYLVIa,, 1958; SZIRMA~ and BALAZS, 1958 ; ROSWNBAUM and D ~ A ~ , 1959 ; DIEZEL and N~IMANIS, 1959; GRAVMA~ and Musso, 1959; PEARSE, 1960; LISO~, 1960; BARKA and A~DERSON, 1963; SI~OH, 1963; CURRA~, 1964). The prevailing opinion concerning the underlying mechanism is that tissue macro-molecules containing electronega- t i re groups in a certain spatial relation, bind the dye cations electrostatically and, under certain conditions, by means of van der Waal forces or hydrogen bonds. Polymerization of the molecules causes the metachromatic shift in absorption maximum.

During recent years, similar studies have been carried out in respect of the Alcian blue staining (Mow~u 1956; BAlbOA and A~I)~soN, 1963; Cm~RAN, 1964; SCOTT, QUINTARI~LLI and DEImOVO, 1964; QUINTa~LLI, SCOTT and D~LLOVO, 1964a and b; LEv and SI'ICE~, 1964). The more exact mechanism must be con- sidered as not yet clarified. By some authors it is characterized as formation of a complex with a salt-like combination of particles of opposite charge (ScoTT, QUINTAt~lgLLI and DELLOVO, 1964), or as being caused by an amidc bond (CvR~A~x, 1964), whereas LlSO~ (1960) preferred the designation "coloration signaletique", until the problem was clarified.

These methods are universally applied when acid mueopolysaeeharides in tissues are to be demonstrated since, as previously mentioned, the pit-dependence of the reactions and the pK-values of the tissue anions concerned are used for the purpose of distinguishing between the various acid mueopolysaeeharides. As stated above, this condition is extremely eompheated by the presence of proteins, because the competitive cationic effect of these substances will often produce a negative metaehromatie reaction at low pH values. Therefore, it was only natural, prior to the metaehromatie reaction, to deaminate the tissue proteins present; this procedure would offer the advantage of maintaining the dye-cation concentration at a constant level throughout the entire pH range instead of being


compelled to increase this concentration at low p H values in order to compete with the protein cations. In particular, such an increase in concentration, as regards the toluidine blue staining, would cause sites with a high concentration of acid mucopolysaccharides (in particular chondroitin sulphate) to be orthochro- matic instead of metachromatic, which might complicate the comparison between various types of tissue. Furthermore, a constant concentration of dye cations would make it possible to assess the degree of metachromasia at decreasing p H values gradually as the various groups of tissue anions (carboxyl, phosphate, sulphate) stop functioning as electronegative groups in consequence of the different p K values of the tissue polyanions. A slight prolongation of the staining t ime in such cases would be sufficient to obtain distinct metachromasia, proportionally as the number of anion groups decreases with decreasing p H values.

The present investigation shows tha t in the tissues examined, the competitive cation effect of the tissue proteins can easily be prevented by oxidative deamination with ninhydrin for 30 min. The prevailing opinion is that ninhydrin produces an oxidative deamination of protein-bound amino acid groups (YASUMA and ICHI- KAWA, 1953; BURSTONE, 1959; KASTEN, 1962; BARKA and ANDV.RSON, 1963). During this deamination an cr acid group is first formed, which is decomposed to an aldehyde giving off ammonia and carbon dioxide. I n order to s tudy this production of aldehyde, control experiments with 10% egg albumin as test film were carried out. These experiments showed tha t t rea tment with ninhydrin for 30 rain did not produce aldehyde groups which could be demonstrated by means of subsequent t rea tment with Schiff's reagent. However, these groups could be demonstrated after deamination with ninhydrin for 2 hours.

The choice of the two types of tissue, cornea and aorta, is based on the fact that in both tissues a large quant i ty of protein is found as compared with the quanti ty of acid mucopolysaccharides and consequently, it must be expected that the competitive cation effect of the proteins will be very pronounced when metachromatie staining at low p H values is carried out. Thus, as regards the cornea, the following wet weight percentages are stated: 13.5% of collagen, 6.5% of non-collagen protein, and 1% of mucopolysaccharide (]~][AURICE, 1962).

Applying various methods, the percentage of acid mucopolysaecharides in the corneal s t roma was found to be 1.8% (MEYER, LINKER, DAVIDSON and WEISS- MANN, 1953) and 4---4.5% of the dry weight {ANsETH, 1961). Almost 50% of the acid mucopolysaecharides was found to be keratosulphate {keratan-), and the remaining 50% consisted of chondroitin sulphate A and chondroitin (equal amounts) (MEYER, LINKER, DAVIDSON and WEISSMANN, 1953; POLATNIK, LA T]~ssA and KATZlN, 1957), whereas ANSV, TH (1961) found approximately two thirds keratosulphate and one third chondroitin sulphate. This may explain why the authors of the present paper found only a slight reduction in the metachromatie reaction after digestion with hyaluronidase, since keratosulphate is not digested by this enzyme.

I t would be expected tha t the corneal s troma would exhibit metaehromasia at the lowest pH values in consequence of the electronegative sulphate groups, but SZmMAI and B~_LAZS (1958) stated tha t the corneal s troma does not take up cationic dyes below a p H of 4.0 and these investigators concluded tha t this was due to the fact tha t the sulphate groups are not free to react a t these p H

260 tt. ANDERSEN and N. E~LERS:

values. This s tatement is in accordance with the observations made by the present authors in connexion with metachromatic staining without previous t reatment with ninhydrin and must be ascribed to the competitive effect of protein cations. In this connexion it is of interest that POLATNIK, LA TESSA and KATZIN (1957a and b) found a content of non-collagen protein in the corneal stroma almost corresponding to the quanti ty of mucopolysaccharides present. Several factors support the assumption tha t the metachromatic reaction is inhibited by proteins in the corneal stroma. This applies to the oxidative deamination of the proteins by means of ninhydrin and to deamination with nitrous acid. To this should be added the similar effect obtained by pepsin digestion of the corneal stroma. Of these three procedures the ninhydrin deamination exerts the maximum morphological preservative effect, since no morphological differences have been observed between tissues treated with ninhydrin and tissues which have not undergone such treatment. Conversely, nitrous acid deamination and pepsin digestion produce certain morphological alterations, which are seen in particular in connexion with pepsin digestion, where inter alia the corneal epithelium disappears completely when the Bowman membrane is digested. Furthermore, the fact that an increase in the concentration of dye cations is able to inhibit the competitive effect of protein cations, points in the same direction and corresponds to experiments with methylene blue staining (KERN and GRANT, 1961).

In the present study the cytoplasmatic granules in the cells of the corneal stroma have been disregarded; these granules, according to KITANO (1966), should consist of keratosulphate.

As regards the aorta, biochemical and histochemical investigations have revealed the presence of chondroitin sulphate A, B, and C, hyaluronic acid (DYR- BYE, 1959; BERTELSEN and J E ~ S ~ , 1960; ZUGIBE, 1962; BRIMACOMBE and WEBBER, 1964), and sialomucoprotein (BARNES, 1965), which are present in addition to the normally appearing elastin (GOTTE, MENEGHELLI and CASTELLANI, 1965). The relationship between the various acid mucopolysaccharides changes according to species and with ageing. Thus, chondroitin sulphate A and C and hyaluronic acid are predominant in young individuals, whereas the quantity of chondroitin sulphate B increases with age.

As is the case in the cornea, a pronounced metachromasia at pH 1.0 should be expected in the aorta, but the experiments showed that this reaction was negative in the frozen sections and only slightly positive in the paraffin sections. As regards both types of sections it applied that ninhydrin t reatment produced marked metachromasia, almost corresponding to that seen after staining with 0.1% toluidine blue in 30% ethanol. The difference observed between frozen sections not treated with ninhydrin and corresponding paraffin sections is believed to be caused by the effect of ethanol during the dehydration prior to the embedd- ing in paraffin, since it was possible to produce the same faint metachromasia at pH 1.0 in frozen sections by treating these sections with absolute ethanol for 3 min before staining. Whether this effect of ethanol is a coagulation of the tissue proteins or a precipitating effect on the quarternary ammonium complexes formed by the cetylpyridinium chloride and the tissue chromotropes must await further studies. A similar effect of ethanol could not be produced in the corneal stroma. As regards the aorta, staining with toluidine blue and Alcian blue showed

Proteins in the Metaehromatic Staining 261

fairly substantial agreement, which applied also to the sections treated with ninhydrin, whereas this was not observed in the corneae where simultaneous treatment of the sections with ninhydrin did not produce any distinct effect on the subsequent Alcian blue staining. As long as the chemical data underlying the Alcian blue staining are not fully clarified, it would not be possible to offer a satisfactory explanation of this difference.

As was to be expected, ninhydrin treatment of the foetal tissue did not exert any influence on the subsequent metachromatic staining at varying values of pH, since at the early stages of development the quantity of acid mucopolysaccharides is very high as compared with the protein present and consequently, the competitive protein cation effect can almost be discoufited.

A comparison between frozen sections and ordinary paraffin sections of all the types of tissue studied showed that the frozen sections exhibited a more homogenous distribution of the acid mucopolysaccharides than that seen in the corresponding paraffin sections, where a certain fibrillar flocculation of the chromotropes prevailed. There is little doubt that the frozen sections give a far more correct picture of the prevailing histological conditions and should be preferred in studies of the localization in the tissue of the acid mucopolysaccharides.

The /ollowing technique may be recommended in studies of tissue polyanions where a competitive effect of protein cations can be expected:

1. Cetylpyridinium chloride-formalin fixation or fixation with buffered neutral 4% formaldehyde.

2. Frozen sections [with dh'ect rinsing in distilled water (low ionic strength)]. 3. Air-drying of sections. 4.0.5% ninhydrin in absolute ethanol for 30 rain at 370 C. 5. Rinsing in tap water for 2--3 min. 6. Toluidine blue staining (0.1%) in varying buffer solutions (pit varying

from 1.0 to 7.0) (staining time depending on the chromotrope concentration). 7. Rinsing in two shifts of distilled water. 8. Dehydration in two shifts of absolute ethanol (3--4 min) (if necessary

preceded by passage through 70 % ethanol). 9. Xylen and mounting in DePex.

This work has been supported by grants from the Danish State Research Foundation.

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Dr. H. ANDERSEN Laboratory of Cyto- and Histochemistry, Dpt. of Human Anatomy (Institute A) University of Copenhagen Copenhagen O, Universitetsparken 1, Denmark

Deamination to prevent the competitive effect of proteins in the metachromatic staining of sections...

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