observations on histological methods involving the use of...

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Observations on histological methods involvingthe use of phosphotungstic and phosphomolybdicacids, with particular reference to staining with

phosphotungstic acid / haematoxylin

By D. BULMER(From the Anatomy Department, The University, Manchester 13)

SummaryAn attempt has been made to elucidate some of the factors involved in differentialstaining of tissues with Mallory's phosphotungstic acid / haematoxylin, and to findout how far these factors are applicable to other histological methods in which phos-photungstic or phosphomolybdic acid is used. Sections have been subjected tovarious pretreatments to find whether specific chemical groupings are responsible forany of the staining reactions, but the results obtained are not always easy to interpret.

The most important single factor in determining the staining reaction of a tissuematerial with PTAH appears to be its relative permeability to the two colour-complexesof the PTAH mixture. The red complex, of larger molecular size, penetrates collagen;muscle-fibres are penetrated mainly by the smaller blue complex, while red blood-corpuscles fixed with formaldehyde are not penetrated by either. The stainingreactions of muscle and red blood-corpuscles can be altered by a methylation pro-cedure, by treatment with performic acid or formic acid, or by mild alkaline hydro-lysis ; but this appears to be due to alteration of permeability rather than to chemicalalteration of any specific dye-binding groups. The effects of blocking reactionsindicate that the binding of both complexes is to basic groups in the tissues, though itis possible that hydroxyl groups and carboxyl groups may also be involved.

It appears that similar factors control the differential staining with other techniqueswhich involve the use of complex acids (Baker, 1958), and that the chemical speci-ficities which have been claimed for some of them are not well founded.

Introduction

D E S P I T E the extensive use of phosphotungstic and phosphomolybdic acidsin histological practice there has been little agreement on the mechanism ofthe several methods in which they are employed. Baker (1958), referring tothe publications of earlier workers, considered that differential staining withacid dyes, as in trichrome methods, is dependent upon differences in thepermeability of proteins to dye molecules of differing molecular sizes, but otherrecent workers appear to have largely ignored this viewpoint. Thus, Monneand Slautterback (1951), using the Azan technique on sea-urchin eggs, sug-gested that the aniline blue was bound by the amino-sugars of mucopoly-saccharides and the azocarmine and orange G by the amino-groups ofproteins. Hrsel (1957) introduced a staining technique employing treatmentwith phosphomolybdic acid, eosin, and light green, after previous mordantingof the section with chromic acid. From the use of blocking techniques hededuced that the light green stained protein-bound amino-groups while the[Quart. J. micr. Sci., Vol. 103, pt. 3, pp. 311-23, 1962.]

312 Buhner—Phosphotungstic acid j haematoxylin

eosin was specific for proteins containing tryptophane. Jones (i960), however,made an observation which appears to support Baker's view. He found thattanned proteins stained with the orange G of Mallory's phosphotungsticacid / aniline blue / orange G mixture. After destruction of the tanning bondswith diaphanol the orange G stainability was replaced by staining with theaniline blue, but this process could be reversed by re-tanning the proteinwith quinone.

Landing, Uzman, and Whipple (1952) described the use of phospho-molybdic acid in a histochemical method for the demonstration of choline.They believed that the complex acid was bound to choline residues in thetissues, and could subsequently be demonstrated in situ by reduction tomolybdenum blue. Treatment of formalin-fixed frozen sections with variousreagents increased the number of structures binding phosphomolybdic acid,and Landing and his colleagues supposed that this was due to the freeing ofbound choline, which had previously been unable to react. Pearse (i960),quoting the method, comments on the increased number of reacting sites inparaffin sections compared with frozen sections, but does not appear to acceptthat these are necessarily associated with the presence of choline.

The reaction of phosphomolybdic acid with collagen was investigated byPuchtler and Isler (1958). They found that the binding of the complex acid,which they demonstrated by the molybdenum blue reaction, resulted in anintense basiphilia of the collagen. They believed that the phosphomolybdicacid became bound to basic groups of the tissue proteins, leaving several ofits own acidic groups free for staining with basic dye, and that in trichromemethods the amphionic aniline blue acts as a basic dye and is held by the freeacidic groups of bound complex acid.

A related problem which has received little recent attention is that of theso-called metachromatic staining produced by Mallory's phosphotungsticacid / haematoxylin mixture. Tissues which take the fibre stain of a trichromemethod are usually red in a phosphotungstic acid / haematoxylin preparation,while those which take the plasma stain of a trichrome method are usuallyblue. This paper records an attempt to elucidate some of the factors whichmay be concerned in the differential staining with phosphotungstic acid / hae-matoxylin and to find how far these are also applicable to other stainingmethods which employ the complex acids.

Methods and resultsThe phosphotungstic acid / haematoxylin mixture (PTAH) contains 2%

phosphotungstic acid (PTA) and o-i% haematoxylin. It is ripened eithernaturally or, as in the present investigation, by the addition of 17-7 mg % ofpotassium permanganate. Mallory's technique (Mallory and Parker, 1929)prescribes fixation in Zenker's fluid and removal of mercury from the sectionwith alcoholic iodine. The section is then exposed to 0.5% permanganate for5-10 min and 5% oxalic acid for 10-20 min(reduced by Lillie(i954)to 5 min),stained in the PTAH mixture for several hours, and taken straight to 95 %

Buhner—Phosphotungstic acid j haematoxylin 313

alcohol and dehydrated, cleared, and mounted. It has been pointed out byPeers (1941) that formalin-fixed sections produce satisfactory results afterpreliminary mordanting with mercuric chloride, while Earle suggested (Lillie,1954) that mercurial treatment could be omitted.

Sections of rat uterus were employed as test material and blocks werefixed in 4% neutral formaldehyde or in pure acetone for 24 h. Formalin-fixedsections of rat striated muscle and tendon were also used. Adequate stainingwas obtained in the formalin-fixed sections without any pretreatment, thoughsmooth muscle tended to have a deep purple rather than a pure blue colour.Nucleoli were stained intensely blue, while the rest of the nuclear materialwas sometimes blue and sometimes red. This may correspond with thedifferent staining of whole and sectioned nuclei with a modified Azan tech-nique (Lison, 1955). The purple staining reaction of smooth muscle appearsto be due to the presence of both blue and red colours, and it is interestingthat the blue binds much more slowly than the red. After a few minutes inthe staining mixture collagen is stained strongly red and muscle less markedlyso—only after about 15 min does the blue stain become more prominent inmuscle.

Mordanting in a saturated solution of mercuric chloride at 5 8° C for 3 h(Peers, 1941), followed byremoval of the mercury, intensified the blue stainingof the muscle-fibres and the staining of collagen, though the latter becomeorange rather than red. Treatment with permanganate and oxalic acid afterthe mercurial mordanting, as prescribed by Mallory and Parker (1929),reduced the staining to a level rather less than that obtained in the untreatedformalin-fixed sections. Treatment of the formalin-fixed sections with per-manganate and oxalic acid, without previous mercurial mordanting, producedweak staining of collagen and patchy, irregular blue staining of muscle. Therewas no apparent difference in the behaviour of formalin-fixed and acetone-fixed sections, except in the staining of red blood-cells. With formalin fixationthe majority of the red blood corpuscles were unstained, while in the acetone-fixed material they were a deep blue.

The PTAH mixture continues to ripen further for several months afterthe initial artificial oxidation. With an old mixture the staining of formalin-fixed sections is intense, with a deep blue colour in the muscle-fibres. Mer-curial mordanting has much less effect on the staining of muscle than with afresh PTAH mixture, and red cells usually stain blue without any pretreat-ment.

In an attempt to elucidate the mechanism of staining, sections were sub-mitted to a variety of pretreatments before exposure to PTAH. For thispurpose formalin-fixed sections were usually employed, without mercurialmordanting or permanganate-oxalic acid treatment, and staining was carriedout in a PTAH mixture within one month of artificial ripening.

Deamination. Treatment with van Slyke's reagent for 24 h before exposurefor several hours to PTAH produced slight reduction in the staining of


collagen and rather more marked impairment in the staining of muscle. Themuscle was, however, a pure blue, in contrast to the purple of the control.When a nitrosated section was treated for a short period with PTAH (about30 min) and compared with a control section exposed to PTAH for the sameperiod there was complete abolition of staining in the muscle while the stain-ing of collagen was only slightly impaired. There are various possible ex-planations of this late appearance of muscle staining after nitrosation. Inparticular, the effect of nitrous acid on some basic groups may be in partreversible by exposure to the staining mixture, or the groups which still bindthe blue stain maybe ones that are resistant to nitrosation but in which stainingis normally delayed.

Treatment with 0-4% ninhydrin for 8 h at 8o° C (Monne and Slautterback,1951) effectively abolished the staining of muscle and nuclei with PTAH.The staining of collagen was very much impaired, and of a purplish tinge.These effects, however, were probably not those of a straightforward oxidativedeamination, since the interposition of mild alkaline hydrolysis after theninhydrin treatment produced red staining of both collagen and muscle.It is likely that ninhydrin has a tanning action, as described by Speakman(1955), and that the change in the staining picture after alkaline treatment isdue partly to hydrolysis of some of the bonds involved in the tanning, andpartly to the usual effect of alkaline pretreatment on staining with PTAH(see below).

Benzoylation. Treatment with 10% benzoyl chloride in pyridine for 24 hcompletely abolished the staining of muscle. The red staining of collagenwas very much reduced, while nuclear staining was apparently unaffected.It is interesting that the resistance to benzoylation resembles that occurring inassociation with the coupled tetrazonium reaction (Pearse, i960).

Acetylation. When sections were treated for 24 h with 40% acetic anhyd-ride in pyridine and exposed for the usual period of several hours to PTAH,the staining of muscle-fibres was strong, with a pure blue colour instead ofthe usual purple shade. The staining of other tissues was unaltered, exceptfor a change in the staining reaction of the basement membrane of the uterineepithelium from red to blue. If, however, the sections were examined after ashort period in the staining mixture and compared with untreated controlsstained for the same period, it was seen that muscle, nuclei, and basementmembrane were unstained in the acetylated sections but quite strongly stainedin the controls. The staining of collagen was reduced in the acetylated sections,and its colour purple. It appears that the acetylation is largely reversed byprolonged exposure to the PTAH mixture, though a slight residual effect, pro-ducing the bluer staining of muscle, remains. Controls exposed to pyridinealone did not differ in staining from untreated sections exposed to PTAH.

In an attempt to reverse the O-acetylation and leave the N-acetylationintact, sections which had been acetylated were exposed to mild alkalinehydrolysis. With short exposure to PTAH they gave normal red staining ofcollagen, deep blue staining of the basement membrane and red staining of

Buhner—Phosphotungstic acid / haematoxylin 315

muscle. Efforts to produce a specific N-acetylation, by methods adapted fromthose of Green, Ang, and Lam (1953), were unsuccessful.

Phosphorylation. Results similar to those of acetylation were produced byphosphorylation with phosphorus oxychloride in pyridine or in chloroform.Sections subsequently exposed for a short time to PTAH gave no staining ofmuscle or nuclei, while collagen was distinctly blue. More prolonged treat-ment with PTAH resulted in the normal red staining of collagen with bluestaining of muscle. The findings of protein chemists (Herriott, 1947) suggestthat the phosphorylation will affect both hydroxyl and amino-groups, and thereversal of phosphorylation by the acidity of the PTAH mixture is not un-expected.

Methylation. Sections were treated for 24 h at 60° C with methanol con-taining o-i% hydrochloric acid before exposure to PTAH. The staining ofcollagen was unaltered while muscle-fibres and nuclei were red instead oftheir usual blue. Red blood-corpuscles, which in untreated formalin-fixedsections did not stain, were intensely blue, but the blue staining was convertedto red by more prolonged exposure to the methylating procedure. Thealteration in the staining picture produced by methylation was incompletelyreversed by exposure of the methylated section to 0-5% potassium perman-ganate for 20 min before staining (Lillie, 1954). Since the permanganatetreatment of the usual PTAH technique weakens the staining of muscle, therestoration of the blue staining in a methylated section may possibly be a truedemethylating effect.

Alkaline hydrolysis. Treatment with a weakly alkaline solution (diluteammonia or sodium borate solution) before PTAH produced red staining ofmuscle and intense blue staining of red cells. A similar effect was obtained bytreatment with a 6 M solution of urea (pH about 7-5), but the alteration inmuscle staining was incomplete. Strong lithium bromide solution had noapparent effect on subsequent staining with PTAH.

Performic acid. Performic acid, prepared by the method of Pearse (i960),produced a result similar to that of alkaline hydrolysis, though the alterationin muscle staining was often incomplete. Prolonged exposure to performicacid did not affect the intense blue staining of the red cells, but methylationafter the performic acid treatment induced red staining. Significantly, com-parison with control sections showed that pure formic acid produced resultsidentical with those of the performic acid mixture.

Acid hydrolysis. Exposure to N-hydrochloric acid for 1 h slightly reducedthe staining of muscle and collagen, without altering the colour of either.Impairment of nuclear staining was more pronounced.

Periodic acid. Exposure to a 2% solution for 1 h had no apparent effect.Lipid extraction. Treatment with hot methanol/chloroform for 48 h did not

reduce subsequent staining of any tissue with PTAH, and the deep blue ofmuscle was rather accentuated.

The foregoing results resemble those reported by Hrsel (1957) on the effects


of blocking agents on staining with his technique. Generally, the effects ofblocking agents on red staining with PTAH are similar to their effects on lightgreen staining with Hrsel's technique, and the effects on blue staining withPTAH are similar to those on the staining with eosin. Methylation andalkaline hydrolysis were not included in Hrsel's investigations, and periodicacid, which Hrsel found to abolish staining with eosin, has no correspondingeffect on staining with PTAH.

To extend the comparison further, similar pretreatments to those used withPTAH were employed on sections which were subsequently stained for i hin Mallory's PTA / aniline blue / orange G mixture. The results which wereobtained indicate a close correspondence between red staining with PTAHand staining with the aniline blue of Mallory's mixture, and between eitherblue staining or lack of staining with PTAH and staining with the orange Gof Mallory's mixture. Deamination somewhat reduced the staining of collagenwith Mallory's mixture—much more with fine than with coarse fibres—whilemuscle stained a pure orange instead of the rather greyish orange producedin a control. Benzoylation grossly impaired staining with aniline blue, but be-cause of the overall yellow colour induced by the benzoylation procedure theeffect on staining with orange G was not clear. Phosphorylation and acetylationconsiderably reduced the staining of collagen with aniline blue, while musclewas a pure orange. Methylation or alkaline hydrolysis induced strong stainingof muscle with aniline blue.

An attempt was made to show the binding of phosphotungstic acid totissue sections by means of the tungsten blue reaction, in a way similar to theuse of the molybdenum blue reaction by Landing and his colleagues (1952)and by Puchtler and Isler (1958). This was unsuccessful, because of the lowintensity of the tungsten blue. To some extent, however, the bound phospho-tungstic acid could be demonstrated indirectly by the increased basiphiliaresulting from its binding to the tissues, comparable with the basiphiliaassociated with the binding of phosphomolybdic acid (PMA) described byPuchtler and Isler. Sections were therefore exposed to 2% PMA for 5 min,and subsequently treated either with acid stannous chloride to produce themolybdenum blue reaction or with a basic thiazine dye to demonstrate thealteration in basiphilia. Sections exposed for 5 min to 2% PTA were treatedonly with the basic dye. PTA and PMA produced similar changes in basi-philia of various tissue structures, so that it may be justifiable to use the dis-tribution of the molybdenum blue reaction after PMA treatment as anindication of the sites at which PTA will also bind. It must be realized, how-ever, that failure to obtain a molybdenum blue reaction in a tissue after PMAtreatment indicates only the absence of bound hexavalent molybdenum(Sidgwick, 1950). PMA treatment may possibly have some effect on a tissueeven when there is no subsequent molybdenum blue reaction.

The results which were obtained by these methods are shown in table 1.Without previous treatment muscle-fibres bound less PMA than collagen,while red cells bound none. The basiphilia of muscle-fibres after PMA or


PTA treatment was less intense than that of collagen, though staining withbasic dye persisted down to very low pH levels. Methylation or alkalinehydrolysis before treatment with complex acid increased the intensity of themolybdenum blue reaction and the induced basiphilia in muscle to a levelsimilar to that obtained in collagen. This appears to confirm the view ofPuchtler and Isler that the basiphilia after treatment with PMA is due tofree acid groups of the complex acid itself and not to tissue acid groupsreleased by binding of the complex acid to basic groups.

TABLE I

Molybdenum blue afterPMA

Basic dye after PMA orPTA

Methylation before PMAtreatment and molyb-denum blue reaction

Deamination before PMAtreatment and molyb-denum blue reaction

Acetylation before PMAtreatment and molyb-denum blue reaction

Collagen

+ + +

+ + +

+ + +

+ +or +

+

Muscle

+

+

+ + +

0

0

Nuclei

(+)

(+)or O

+ + +

+

+

Red blood-corpuscles

0

O

+ -T

0

0

+ + + denotes a very strong reaction, + + and + reactions of less intensity. ( + ) indicatesa very faint reaction, probably inconsistent from one part of the section to another, and O the

complete absence of any reaction.

Because of the employment of mercuric chloride as mordant in the PTAHtechnique and of chromium trioxide in the method of Hrsel (1957), the effectsof these two reagents were investigated further. It has been pointed out thatmercuric chloride increases the staining intensity of both muscle and collagenwith PTAH, particularly intensifying the blue colour in muscle. In addition,if a section which has been methylated or subjected to alkaline hydrolysis orperformic acid treatment is exposed to mercuric chloride before staining withPTAH, the normal blue staining of muscle is restored. If, on the other hand,the pretreatment is carried out after the mercurial mordanting, the musclestains red. Treatment of a section with mercuric chloride before submittingit to PMA increases the intensity of the resulting basiphilia and molybdenumblue reaction in collagen, but greatly reduces their intensity in muscle.

Similar effects are obtained when chromium trioxide is used in place ofmercuric chloride, and this appears to be the basis of the mordanting inHrsel's method. If the chromium trioxide is omitted methylated muscle-fibres stain with the light green of Hrsel's method, but if the methylation isfollowed by treatment with chromium trioxide the normal staining with eosinis restored. A similar result occurs after performic acid treatment, which,


without chromium mordanting, converts the red staining of muscle to green.The effect of performic acid on staining with Hrsel's method is, like its effecton PTAH staining, reproduced by treatment with formic acid alone, and thiscasts some doubt on Hrsel's conclusion that the effect of performic acid treat-ment is due to a specific destruction of tryptophane. Moreover, in a sectionwhich has been exposed to performic acid for twice the period necessary forcomplete abolition of the DMAB-nitrite reaction (Adams, 1957), the eosinstaining of muscle with Hrsel's method is restored by mordanting withchromium trioxide or mercuric chloride.

In an attempt to shed further light on the staining mechanism with PTAH,a study was made of the staining mixture itself. PTAH is deep purple andhas a pH of about 2-5. Addition of strong acid turns it bright red; addition ofalkali first turns it blue, but with further alkali the dye lake may be broken.Addition of glycine to PTAH produces little change in colour; tryptophane,and to a less marked extent serine, change the colour towards blue, whilelysine and histidine induce the formation of a deep blue precipitate. Thereactions of the latter two amino-acids are presumably associated with theirbasicity, while the reaction with tryptophane may be due to reduction of thePTA, in a comparable manner to the reduction of Folin's phenol reagent bytryptophane (Herriott, 1947).

If the PTAH mixture is boiled it becomes light red, but the original purplecolour is restored on cooling. Paper chromatography, with water or diluteacetic acid as dispersion medium, distinguishes a more rapidly diffusing bluecolour from a more slowly diffusing red. This is comparable with chromato-graphy of Mallory's PTA / aniline blue / orange G mixture, where the rapidlydiffusing orange is separated from the more slowly diffusing blue. Electro-phoresis of PTAH through an agar gel, as described by Baker (1958), showsthat both its red and blue components are anionic.

Sections stained with PTAH are extremely fast to treatment with strongacid. Dilute alkali rapidly elutes the stain, more rapidly with the red than theblue, while a 6 M solution of urea removes the red but leaves the blue intact.This may be compared with the behaviour of sections stained with anilineblue. Used as a 1% solution in acetic acid, aniline blue stains all tissues buthas a particularly strong affinity for collagen. The stain is eluted by 6 M ureamuch more rapidly than by alkaline solutions of similar pH. In conjunctionwith either PMA or PTA, staining with aniline blue is slower and less intense,but the dye is now bound specifically to such structures as collagen. Herealso it is rapidly eluted by urea solution, though the complex acid remainsbound to the tissues and can be demonstrated by the molybdenum bluereaction or by the increase of basiphilia.

Differential staining with PTAH is dependent upon the relative proportionsof PTA and haematein in the staining mixture. If a mixture is made containingten times the usual proportion of haematoxylin to PTA, comparable in con-stitution to the phosphomolybdic acid-haematoxylin mixture of Mallory, it is


of a deep, bluish purple colour which, like PMAH, stains all tissue componentsthe same colour. On the other hand, a great increase in the proportion of PTAresults in a light red mixture, which colours tissue sections only very faintly.

DiscussionThe results indicate that the different staining methods which have been

investigated are fundamentally similar. Tissues such as red cells, which donot bind the complex acids in a demonstrable form in formalin-fixed paraffinsections, do not stain with PTAH and stain only with the eosin of Hrsel'stechnique and the orange G of Mallory's PTA / aniline blue / orange Gmixture. Collagen, which binds the complex acids strongly, stains red withPTAH and stains with the light green of HrSel's technique and the anilineblue of Mallory's mixture. Muscle-fibres and nucleoli bind the complex acidsless strongly than collagen, stain blue with PTAH and take the eosin of Hrsel'stechnique and the orange G of Mallory's mixture. With all the methods,however, muscle tends to take an element of the fibre stain unless some pre-treatment, such as mercurial or chromium mordanting, is employed. Methy-lation or treatment with dilute alkali makes the staining of muscle resemblethe normal staining of collagen unless mercurial or chromium mordanting isinterposed. Alkaline hydrolysis makes the staining of red cells resemble thatof untreated muscle-fibres, while prolonged methylation makes them stainlike collagen fibres.

The hypotheses suggested by other workers will be considered first. Thereis no evidence to support the view of Monne and Slautterback (1951) that thefibre stain of trichrome methods binds to the amino-groups of amino-sugarsand the plasma stain to protein-bound amino-groups, other than the fact thatcertain mucoprotein materials do take the fibre stain. Although it is impossibleto refute this hypothesis completely, because of the lack of specific histo-chemical reactions for amino-sugars, the effects of mordanting and blockingtechniques are difficult to reconcile with it.

The opinion of Landing and his colleagues (1952), that PMA reacts speci-fically with choline, does not appear to be justifiable, as there are many othertissue constituents with which the complex acids might theoretically be ex-pected to react. Lipid extraction does not affect subsequent staining withPTAH.

The results reported by Puchtler and Isler (1958) on the tissue binding ofPMA have been largely confirmed by the present investigation, though, aswill be pointed out later, their explanation of the staining of collagen withaniline blue in the presence of complex acid is not the only one to fit the avail-able facts.

The view of Hrsel, who considered that eosin staining with his technique isassociated with the presence of tryptophane, merits some consideration. Itmight similarly be suggested that blue staining with PTAH indicates thepresence of protein-bound tryptophane, though proteins that contain trypto-phane may require pretreatment with alkali, as with red cells, or potassium


dichromate, as with fibrin (Pearse, 1960), before staining blue. Such an ex-planation did indeed present itself early in this investigation, when the effectof pretreatment with performic acid was first noticed. The similar effect offormic acid alone, however, together with the restoration of the blue stainingof muscle by mercurial or chromium mordanting, indicates that the alterationof staining after performic acid is not due to a specific destruction of trypto-phane. The production of a blue colour when tryptophane is added to thePTAH mixture and the known reduction of Folin's reagent by tryptophanesuggest that there may be a characteristic reaction with PTAH, but this isinsufficient basis for a hypothesis. Moreover, Hrsel himself pointed out ex-ceptions to his rule, mentioning proteins that contain tryptophane but stainwith the light green of his technique.

The most satisfactory explanation of the differential staining with PTAHand of its alteration by the various pretreatments which have been employedin this investigation appears to be that suggested by Baker (1958) for stainingwith acid dyes generally. Thus, the red PTAH complex seems to be of largermolecular size than the blue complex and, correspondingly, the aniline blue ofMallory's mixture is of larger molecular size than the orange G. Collagen ispermeated by the largest molecules, the red PTAH and the aniline blue, andmuscle by the smaller blue PTAH complex and the orange G. Red cells arepermeated ohly by the orange G, which is presumably of still smaller molecularsize than the blue PTAH complex. The effects of some of the pretreatmentswhich were employed may be due simply to interference with the configura-tion of the protein molecule rather than to removal of any specific reactinggroup. Alkaline hydrolysis, for instance, appears to 'open up' the muscleprotein sufficiently for the entry of the largest molecules, and the red cellprotein sufficiently for the entry of the intermediate blue PTAH complex.The effect of methylation may possibly have a similar basis. The actions ofchromium mordanting in preventing the element of fibre stain in muscle andin restoring normal staining of muscle after methylation or alkaline hydrolysismay be due to the formation of cross-linkages in the tissue protein, reducingits permeability. On the other hand, mercurial mordanting has the sameeffects on the staining picture, though mercury is said not to form cross-linkages (Pearse, i960). However, the reactions of mercuric chloride withtissue proteins are far from clear (Baker, 1958).

It might be expected that if the configuration of a protein permits the entryof a particular dye or dye complex, certain specific groups which will bind thedye must also be present for staining to occur. The results of the blockingtechniques give no very firm evidence of what groups are involved in thebinding of the different dyes. The failure of nitrosation to have any signi-ficant effect on the staining of the thicker collagen fibres suggests the parti-cipation there of groups other than primary and secondary amines. The verygross impairment in the staining of collagen produced by benzoylation indi-cates that other basic groups and hydroxyl groups may be involved, though,as Lillie (1958) points out, the effects of benzoylation seem to be more far-

Buhner—Phosphotungstic acid j haematoxylin 321

reaching than a simple blockade of basic and hydroxyl groups. There does,however, seem to be some variation between different tissues in their res-ponses to blocking techniques. Thus, the staining of fine collagen fibres isconsiderably reduced by nitrosation, while Monne and Slautterback (1951)found that deamination completely abolished aniline-blue staining in the yolkof sea-urchin eggs. It is possible that amino-groups and other groups arecapable of binding the red PTAH complex or the aniline blue of Mallory'smixture, but that in fine collagen fibres and in the yolk of sea-urchin eggs theamino-groups predominate while in thicker collagen fibres other groups aremore important. Alternatively, and much more probably, the basic groupsof the thick collagen fibres may simply be more resistant to nitrosation, eitherfor physical or chemical reasons. It has been pointed out by Terner andClark (i960) that substitution in amino-groups may make them resistant tonitrosation, though they continue to bind acid dyes.

The hydroxyl groups of hydroxy-proline may possibly be involved in thebinding of the red PTAH complex to collagen, but the elution of the PTAHby strong urea solution is hardly sufficient evidence to suggest that hydrogenbonding is concerned in this when mild alkaline hydrolysis is equally effective.However, the rapid elution of aniline blue by urea solution suggests thathydrogen bonding may be involved in the binding of aniline blue to collagen.Gustavson (1957) has considered that the amphionic dye benzopurpurine 4Bis held by hydrogen bonding to the hydroxyl groups of collagen, and a similarexplanation may account for the great affinity of aniline blue for collagen.Puchtler and Isler (1958) explained the binding of aniline blue to collagen inthe presence of PMA by suggesting that the dye was held by free acidic groupsof tissue-bound complex acid. There is no direct evidence that this is true, orthat the reduction in the staining of collagen by aniline blue when the dye isused in conjunction with complex acid instead of in simple acid solution isnot due to competition for binding sites between dye and polyacid (Baker,1958).

The failure of muscle to stain with short exposure to PTAH after acety-lation, phosphorylation and deamination implies that amino-groups andprobably other basic groups and hydroxyl groups are involved in the bindingof the blue PTAH complex. The conversion of the blue staining to red bymethylation or alkaline hydrolysis may be due, as has already been suggested,to rupture of intramolecular bonds in the muscle protein. Obviously methy-lation, which has been claimed to be specific for carboxyl groups (Fraenkel-Conrat and Olcott, 1945), would be expected to affect linkages betweencarboxyl groups and other groups. The very mild and brief alkaline hydrolysismay break ionic linkages, and the similar though less complete effect of ureasolution suggests that rupture of hydrogen bonds may be involved in thealteration of the staining reaction of muscle. On the other hand, it is possiblethat carboxyl groups are necessary for the binding of the blue complex tomuscle, and this is suggested by the partial restoration of blue staini ng afterpermanganate 'demethylation'. One would have to suppose, then, t hat the

322 Buhner—Phosphotungstic acid / haematoxylin

carboxyl groups of red cells are less easily methylated than those of muscle.Moreover, if carboxyl groups are essential for the binding of the blue PTAHcomplex, it is difficult to explain the increased blue staining after mercurial orchromium mordanting—which, at any rate according to some authorities(Pearse, i960)—would be expected to block carboxyl groups. There is, how-ever, an indication that acidic groups may be involved in the binding ofcomplex acid to nuclei, in that treatment with PMA or PTA greatly reducesthe normal nuclear basiphilia. After methylation, treatment with complexacid induces a nuclear basiphilia comparable with that occurring in collagen.

The blue and the red staining with PTAH must be due to different poly-acid / dye complexes and in some respects the PTAH mixture resembles thelakes of PTA with basic dyes (Pratt, 1947). Thus an increase in polyacidcontent or heating of the mixture lightens the colour. In addition, the simi-larity of the staining distribution of the red PTAH complex with that of thebasic thiazine dyes after pretreatment of the section with PMA or PTA suggeststhat the red PTAH may be a complex of similar structure to the lakes of PTAwith basic dyes. PTA might be expected to bind to haematein in this way,since haematein will be positively charged when the pH is below its iso-electric point of about 6-5 (Baker, 1958). Possibly the blue complex is ofa similar nature, but with a lower polyacid content and molecular size. Onthe other hand, PTA may form complexes of another type with haematein,similar to the complexes of tungstic acid with polyhydroxy-phenols which,because of their stability, are presumed to contain chelate linkages (Sidgwick,1950). The blue PTAH complex may be of this nature, though it is of courselikely that both red and blue staining are each due to mixtures of severaldifferent complexes.

No complete explanation can be offered for the staining reactions of tissuetonstituents with the various preparations which have been studied, but it isclear that differential staining with all of them is dependent upon similarfundamental principles. The most satisfactory approach appears to be that ofrelating the staining by different dye complexes with the permeability oftissue proteins to molecules of differing sizes, as Baker has described. Tissuegroups must also be available to bind the dye complex once it has permeatedthe protein, but until more specific chemical techniques can be applied it isimpossible to define these precisely. In conclusion it is worth emphasizingthat the results of blocking techniques in an investigation of this kind must beinterpreted with caution. Effects on staining reactions may often be due tointerference with the normal configuration of the protein molecule ratherthan to blockade of a specific reactive group.

My thanks are due to Professor R. D. Lockhart, in whose Department theinitial part of the work was carried out, and to Professor G. A. G. Mitchellfor his valuable advice on the preparation of this paper.


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