Observations on histological methods involving the use of phosphotungstic

311
Observations on histological methods involving
the use of phosphotungstic and phosphomolybdic
acids, with particular reference to staining with
phosphotungstic acid / haematoxylin
By D. BULMER
(From the Anatomy Department, The University, Manchester 13)
Summary
An attempt has been made to elucidate some of the factors involved in differential
staining of tissues with Mallory's phosphotungstic acid / haematoxylin, and to find
out how far these factors are applicable to other histological methods in which phosphotungstic or phosphomolybdic acid is used. Sections have been subjected to
various pretreatments to find whether specific chemical groupings are responsible for
any 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 tissue
material with PTAH appears to be its relative permeability to the two colour-complexes
of the PTAH mixture. The red complex, of larger molecular size, penetrates collagen;
muscle-fibres are penetrated mainly by the smaller blue complex, while red bloodcorpuscles fixed with formaldehyde are not penetrated by either. The staining
reactions of muscle and red blood-corpuscles can be altered by a methylation procedure, by treatment with performic acid or formic acid, or by mild alkaline hydrolysis ; but this appears to be due to alteration of permeability rather than to chemical
alteration of any specific dye-binding groups. The effects of blocking reactions
indicate that the binding of both complexes is to basic groups in the tissues, though it
is possible that hydroxyl groups and carboxyl groups may also be involved.
It appears that similar factors control the differential staining with other techniques
which involve the use of complex acids (Baker, 1958), and that the chemical specificities 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 acids
in histological practice there has been little agreement on the mechanism of
the several methods in which they are employed. Baker (1958), referring to
the publications of earlier workers, considered that differential staining with
acid dyes, as in trichrome methods, is dependent upon differences in the
permeability of proteins to dye molecules of differing molecular sizes, but other
recent workers appear to have largely ignored this viewpoint. Thus, Monne
and Slautterback (1951), using the Azan technique on sea-urchin eggs, suggested that the aniline blue was bound by the amino-sugars of mucopolysaccharides and the azocarmine and orange G by the amino-groups of
proteins. Hrsel (1957) introduced a staining technique employing treatment
with phosphomolybdic acid, eosin, and light green, after previous mordanting
of the section with chromic acid. From the use of blocking techniques he
deduced that the light green stained protein-bound amino-groups while the
[Quart. J. micr. Sci., Vol. 103, pt. 3, pp. 311-23, 1962.]
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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 that
tanned proteins stained with the orange G of Mallory's phosphotungstic
acid / aniline blue / orange G mixture. After destruction of the tanning bonds
with diaphanol the orange G stainability was replaced by staining with the
aniline blue, but this process could be reversed by re-tanning the protein
with quinone.
Landing, Uzman, and Whipple (1952) described the use of phosphomolybdic acid in a histochemical method for the demonstration of choline.
They believed that the complex acid was bound to choline residues in the
tissues, and could subsequently be demonstrated in situ by reduction to
molybdenum blue. Treatment of formalin-fixed frozen sections with various
reagents increased the number of structures binding phosphomolybdic acid,
and Landing and his colleagues supposed that this was due to the freeing of
bound choline, which had previously been unable to react. Pearse (i960),
quoting the method, comments on the increased number of reacting sites in
paraffin sections compared with frozen sections, but does not appear to accept
that these are necessarily associated with the presence of choline.
The reaction of phosphomolybdic acid with collagen was investigated by
Puchtler and Isler (1958). They found that the binding of the complex acid,
which they demonstrated by the molybdenum blue reaction, resulted in an
intense basiphilia of the collagen. They believed that the phosphomolybdic
acid became bound to basic groups of the tissue proteins, leaving several of
its own acidic groups free for staining with basic dye, and that in trichrome
methods the amphionic aniline blue acts as a basic dye and is held by the free
acidic groups of bound complex acid.
A related problem which has received little recent attention is that of the
so-called metachromatic staining produced by Mallory's phosphotungstic
acid / haematoxylin mixture. Tissues which take the fibre stain of a trichrome
method are usually red in a phosphotungstic acid / haematoxylin preparation,
while those which take the plasma stain of a trichrome method are usually
blue. This paper records an attempt to elucidate some of the factors which
may be concerned in the differential staining with phosphotungstic acid / haematoxylin and to find how far these are also applicable to other staining
methods which employ the complex acids.
Methods and results
The phosphotungstic acid / haematoxylin mixture (PTAH) contains 2%
phosphotungstic acid (PTA) and o-i% haematoxylin. It is ripened either
naturally or, as in the present investigation, by the addition of 17-7 mg % of
potassium permanganate. Mallory's technique (Mallory and Parker, 1929)
prescribes fixation in Zenker's fluid and removal of mercury from the section
with alcoholic iodine. The section is then exposed to 0.5% permanganate for
5-10 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 by
Peers (1941) that formalin-fixed sections produce satisfactory results after
preliminary 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 were
fixed in 4% neutral formaldehyde or in pure acetone for 24 h. Formalin-fixed
sections of rat striated muscle and tendon were also used. Adequate staining
was obtained in the formalin-fixed sections without any pretreatment, though
smooth muscle tended to have a deep purple rather than a pure blue colour.
Nucleoli were stained intensely blue, while the rest of the nuclear material
was sometimes blue and sometimes red. This may correspond with the
different staining of whole and sectioned nuclei with a modified Azan technique (Lison, 1955). The purple staining reaction of smooth muscle appears
to be due to the presence of both blue and red colours, and it is interesting
that the blue binds much more slowly than the red. After a few minutes in
the staining mixture collagen is stained strongly red and muscle less markedly
so—only after about 15 min does the blue stain become more prominent in
muscle.
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 staining
of the muscle-fibres and the staining of collagen, though the latter become
orange rather than red. Treatment with permanganate and oxalic acid after
the mercurial mordanting, as prescribed by Mallory and Parker (1929),
reduced the staining to a level rather less than that obtained in the untreated
formalin-fixed sections. Treatment of the formalin-fixed sections with permanganate and oxalic acid, without previous mercurial mordanting, produced
weak staining of collagen and patchy, irregular blue staining of muscle. There
was no apparent difference in the behaviour of formalin-fixed and acetonefixed sections, except in the staining of red blood-cells. With formalin fixation
the majority of the red blood corpuscles were unstained, while in the acetonefixed material they were a deep blue.
The PTAH mixture continues to ripen further for several months after
the initial artificial oxidation. With an old mixture the staining of formalinfixed sections is intense, with a deep blue colour in the muscle-fibres. Mercurial mordanting has much less effect on the staining of muscle than with a
fresh PTAH mixture, and red cells usually stain blue without any pretreatment.
In an attempt to elucidate the mechanism of staining, sections were submitted to a variety of pretreatments before exposure to PTAH. For this
purpose formalin-fixed sections were usually employed, without mercurial
mordanting or permanganate-oxalic acid treatment, and staining was carried
out in a PTAH mixture within one month of artificial ripening.
Deamination. Treatment with van Slyke's reagent for 24 h before exposure
for several hours to PTAH produced slight reduction in the staining of
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collagen and rather more marked impairment in the staining of muscle. The
muscle 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 (about
30 min) and compared with a control section exposed to PTAH for the same
period there was complete abolition of staining in the muscle while the staining of collagen was only slightly impaired. There are various possible explanations of this late appearance of muscle staining after nitrosation. In
particular, the effect of nitrous acid on some basic groups may be in part
reversible by exposure to the staining mixture, or the groups which still bind
the blue stain maybe ones that are resistant to nitrosation but in which staining
is 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 oxidative
deamination, since the interposition of mild alkaline hydrolysis after the
ninhydrin 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 is
due partly to hydrolysis of some of the bonds involved in the tanning, and
partly to the usual effect of alkaline pretreatment on staining with PTAH
(see below).
Benzoylation. Treatment with 10% benzoyl chloride in pyridine for 24 h
completely abolished the staining of muscle. The red staining of collagen
was very much reduced, while nuclear staining was apparently unaffected.
It is interesting that the resistance to benzoylation resembles that occurring in
association with the coupled tetrazonium reaction (Pearse, i960).
Acetylation. When sections were treated for 24 h with 40% acetic anhydride 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 of
the usual purple shade. The staining of other tissues was unaltered, except
for a change in the staining reaction of the basement membrane of the uterine
epithelium from red to blue. If, however, the sections were examined after a
short period in the staining mixture and compared with untreated controls
stained for the same period, it was seen that muscle, nuclei, and basement
membrane were unstained in the acetylated sections but quite strongly stained
in the controls. The staining of collagen was reduced in the acetylated sections,
and its colour purple. It appears that the acetylation is largely reversed by
prolonged exposure to the PTAH mixture, though a slight residual effect, producing the bluer staining of muscle, remains. Controls exposed to pyridine
alone did not differ in staining from untreated sections exposed to PTAH.
In an attempt to reverse the O-acetylation and leave the N-acetylation
intact, sections which had been acetylated were exposed to mild alkaline
hydrolysis. With short exposure to PTAH they gave normal red staining of
collagen, 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 from
those of Green, Ang, and Lam (1953), were unsuccessful.
Phosphorylation. Results similar to those of acetylation were produced by
phosphorylation with phosphorus oxychloride in pyridine or in chloroform.
Sections subsequently exposed for a short time to PTAH gave no staining of
muscle or nuclei, while collagen was distinctly blue. More prolonged treatment with PTAH resulted in the normal red staining of collagen with blue
staining of muscle. The findings of protein chemists (Herriott, 1947) suggest
that the phosphorylation will affect both hydroxyl and amino-groups, and the
reversal of phosphorylation by the acidity of the PTAH mixture is not unexpected.
Methylation. Sections were treated for 24 h at 60° C with methanol containing o-i% hydrochloric acid before exposure to PTAH. The staining of
collagen was unaltered while muscle-fibres and nuclei were red instead of
their usual blue. Red blood-corpuscles, which in untreated formalin-fixed
sections did not stain, were intensely blue, but the blue staining was converted
to red by more prolonged exposure to the methylating procedure. The
alteration in the staining picture produced by methylation was incompletely
reversed by exposure of the methylated section to 0-5% potassium permanganate for 20 min before staining (Lillie, 1954). Since the permanganate
treatment of the usual PTAH technique weakens the staining of muscle, the
restoration of the blue staining in a methylated section may possibly be a true
demethylating effect.
Alkaline hydrolysis. Treatment with a weakly alkaline solution (dilute
ammonia or sodium borate solution) before PTAH produced red staining of
muscle and intense blue staining of red cells. A similar effect was obtained by
treatment with a 6 M solution of urea (pH about 7-5), but the alteration in
muscle staining was incomplete. Strong lithium bromide solution had no
apparent 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 alteration
in muscle staining was often incomplete. Prolonged exposure to performic
acid did not affect the intense blue staining of the red cells, but methylation
after the performic acid treatment induced red staining. Significantly, comparison with control sections showed that pure formic acid produced results
identical with those of the performic acid mixture.
Acid hydrolysis. Exposure to N-hydrochloric acid for 1 h slightly reduced
the 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 of
muscle was rather accentuated.
The foregoing results resemble those reported by Hrsel (1957) on the effects
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of blocking agents on staining with his technique. Generally, the effects of
blocking agents on red staining with PTAH are similar to their effects on light
green staining with Hrsel's technique, and the effects on blue staining with
PTAH are similar to those on the staining with eosin. Methylation and
alkaline hydrolysis were not included in Hrsel's investigations, and periodic
acid, which Hrsel found to abolish staining with eosin, has no corresponding
effect on staining with PTAH.
To extend the comparison further, similar pretreatments to those used with
PTAH were employed on sections which were subsequently stained for i h
in Mallory's PTA / aniline blue / orange G mixture. The results which were
obtained indicate a close correspondence between red staining with PTAH
and staining with the aniline blue of Mallory's mixture, and between either
blue staining or lack of staining with PTAH and staining with the orange G
of Mallory's mixture. Deamination somewhat reduced the staining of collagen
with Mallory's mixture—much more with fine than with coarse fibres—while
muscle stained a pure orange instead of the rather greyish orange produced
in a control. Benzoylation grossly impaired staining with aniline blue, but because of the overall yellow colour induced by the benzoylation procedure the
effect on staining with orange G was not clear. Phosphorylation and acetylation
considerably reduced the staining of collagen with aniline blue, while muscle
was a pure orange. Methylation or alkaline hydrolysis induced strong staining
of muscle with aniline blue.
An attempt was made to show the binding of phosphotungstic acid to
tissue sections by means of the tungsten blue reaction, in a way similar to the
use of the molybdenum blue reaction by Landing and his colleagues (1952)
and by Puchtler and Isler (1958). This was unsuccessful, because of the low
intensity of the tungsten blue. To some extent, however, the bound phosphotungstic acid could be demonstrated indirectly by the increased basiphilia
resulting from its binding to the tissues, comparable with the basiphilia
associated with the binding of phosphomolybdic acid (PMA) described by
Puchtler and Isler. Sections were therefore exposed to 2% PMA for 5 min,
and subsequently treated either with acid stannous chloride to produce the
molybdenum blue reaction or with a basic thiazine dye to demonstrate the
alteration in basiphilia. Sections exposed for 5 min to 2% PTA were treated
only with the basic dye. PTA and PMA produced similar changes in basiphilia of various tissue structures, so that it may be justifiable to use the distribution of the molybdenum blue reaction after PMA treatment as an
indication of the sites at which PTA will also bind. It must be realized, however, that failure to obtain a molybdenum blue reaction in a tissue after PMA
treatment indicates only the absence of bound hexavalent molybdenum
(Sidgwick, 1950). PMA treatment may possibly have some effect on a tissue
even 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
Buhner—Phosphotungstic acid / haematoxylin
317
PTA treatment was less intense than that of collagen, though staining with
basic dye persisted down to very low pH levels. Methylation or alkaline
hydrolysis before treatment with complex acid increased the intensity of the
molybdenum blue reaction and the induced basiphilia in muscle to a level
similar to that obtained in collagen. This appears to confirm the view of
Puchtler and Isler that the basiphilia after treatment with PMA is due to
free acid groups of the complex acid itself and not to tissue acid groups
released by binding of the complex acid to basic groups.
TABLE I
Molybdenum blue after
PMA
Basic dye after PMA or
PTA
Methylation before PMA
treatment and molybdenum blue reaction
Deamination before PMA
treatment and molybdenum blue reaction
Acetylation before PMA
treatment and molybdenum blue reaction
Collagen
Muscle
Nuclei
Red bloodcorpuscles
+++
+
(+)
0
+++
+
(+)
O
+++
+++
+++
+ -T
++
or +
+
0
+
0
0
+
0
or O
+ + + denotes a very strong reaction, + + and + reactions of less intensity. ( + ) indicates
a 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 PTAH
technique and of chromium trioxide in the method of Hrsel (1957), the effects
of these two reagents were investigated further. It has been pointed out that
mercuric chloride increases the staining intensity of both muscle and collagen
with PTAH, particularly intensifying the blue colour in muscle. In addition,
if a section which has been methylated or subjected to alkaline hydrolysis or
performic acid treatment is exposed to mercuric chloride before staining with
PTAH, the normal blue staining of muscle is restored. If, on the other hand,
the pretreatment is carried out after the mercurial mordanting, the muscle
stains red. Treatment of a section with mercuric chloride before submitting
it to PMA increases the intensity of the resulting basiphilia and molybdenum
blue reaction in collagen, but greatly reduces their intensity in muscle.
Similar effects are obtained when chromium trioxide is used in place of
mercuric chloride, and this appears to be the basis of the mordanting in
Hrsel's method. If the chromium trioxide is omitted methylated musclefibres stain with the light green of Hrsel's method, but if the methylation is
followed by treatment with chromium trioxide the normal staining with eosin
is restored. A similar result occurs after performic acid treatment, which,
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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 effect
on PTAH staining, reproduced by treatment with formic acid alone, and this
casts some doubt on Hrsel's conclusion that the effect of performic acid treatment is due to a specific destruction of tryptophane. Moreover, in a section
which has been exposed to performic acid for twice the period necessary for
complete abolition of the DMAB-nitrite reaction (Adams, 1957), the eosin
staining of muscle with Hrsel's method is restored by mordanting with
chromium 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 and
has a pH of about 2-5. Addition of strong acid turns it bright red; addition of
alkali 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, while
lysine and histidine induce the formation of a deep blue precipitate. The
reactions of the latter two amino-acids are presumably associated with their
basicity, while the reaction with tryptophane may be due to reduction of the
PTA, in a comparable manner to the reduction of Folin's phenol reagent by
tryptophane (Herriott, 1947).
If the PTAH mixture is boiled it becomes light red, but the original purple
colour is restored on cooling. Paper chromatography, with water or dilute
acetic acid as dispersion medium, distinguishes a more rapidly diffusing blue
colour from a more slowly diffusing red. This is comparable with chromatography of Mallory's PTA / aniline blue / orange G mixture, where the rapidly
diffusing orange is separated from the more slowly diffusing blue. Electrophoresis of PTAH through an agar gel, as described by Baker (1958), shows
that both its red and blue components are anionic.
Sections stained with PTAH are extremely fast to treatment with strong
acid. Dilute alkali rapidly elutes the stain, more rapidly with the red than the
blue, 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 aniline
blue. Used as a 1% solution in acetic acid, aniline blue stains all tissues but
has a particularly strong affinity for collagen. The stain is eluted by 6 M urea
much more rapidly than by alkaline solutions of similar pH. In conjunction
with 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. Here
also it is rapidly eluted by urea solution, though the complex acid remains
bound to the tissues and can be demonstrated by the molybdenum blue
reaction or by the increase of basiphilia.
Differential staining with PTAH is dependent upon the relative proportions
of PTA and haematein in the staining mixture. If a mixture is made containing
ten times the usual proportion of haematoxylin to PTA, comparable in constitution to the phosphomolybdic acid-haematoxylin mixture of Mallory, it is
Buhner—Phosphotungstic acid / haematoxylin
319
of a deep, bluish purple colour which, like PMAH, stains all tissue components
the same colour. On the other hand, a great increase in the proportion of PTA
results in a light red mixture, which colours tissue sections only very faintly.
Discussion
The results indicate that the different staining methods which have been
investigated are fundamentally similar. Tissues such as red cells, which do
not bind the complex acids in a demonstrable form in formalin-fixed paraffin
sections, do not stain with PTAH and stain only with the eosin of Hrsel's
technique and the orange G of Mallory's PTA / aniline blue / orange G
mixture. Collagen, which binds the complex acids strongly, stains red with
PTAH and stains with the light green of HrSel's technique and the aniline
blue of Mallory's mixture. Muscle-fibres and nucleoli bind the complex acids
less strongly than collagen, stain blue with PTAH and take the eosin of Hrsel's
technique 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 pretreatment, such as mercurial or chromium mordanting, is employed. Methylation or treatment with dilute alkali makes the staining of muscle resemble
the normal staining of collagen unless mercurial or chromium mordanting is
interposed. Alkaline hydrolysis makes the staining of red cells resemble that
of untreated muscle-fibres, while prolonged methylation makes them stain
like collagen fibres.
The hypotheses suggested by other workers will be considered first. There
is no evidence to support the view of Monne and Slautterback (1951) that the
fibre stain of trichrome methods binds to the amino-groups of amino-sugars
and the plasma stain to protein-bound amino-groups, other than the fact that
certain mucoprotein materials do take the fibre stain. Although it is impossible
to refute this hypothesis completely, because of the lack of specific histochemical reactions for amino-sugars, the effects of mordanting and blocking
techniques are difficult to reconcile with it.
The opinion of Landing and his colleagues (1952), that PMA reacts specifically with choline, does not appear to be justifiable, as there are many other
tissue constituents with which the complex acids might theoretically be expected to react. Lipid extraction does not affect subsequent staining with
PTAH.
The results reported by Puchtler and Isler (1958) on the tissue binding of
PMA have been largely confirmed by the present investigation, though, as
will be pointed out later, their explanation of the staining of collagen with
aniline blue in the presence of complex acid is not the only one to fit the available facts.
The view of Hrsel, who considered that eosin staining with his technique is
associated with the presence of tryptophane, merits some consideration. It
might similarly be suggested that blue staining with PTAH indicates the
presence of protein-bound tryptophane, though proteins that contain tryptophane may require pretreatment with alkali, as with red cells, or potassium
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dichromate, as with fibrin (Pearse, 1960), before staining blue. Such an explanation did indeed present itself early in this investigation, when the effect
of pretreatment with performic acid was first noticed. The similar effect of
formic acid alone, however, together with the restoration of the blue staining
of muscle by mercurial or chromium mordanting, indicates that the alteration
of staining after performic acid is not due to a specific destruction of tryptophane. The production of a blue colour when tryptophane is added to the
PTAH mixture and the known reduction of Folin's reagent by tryptophane
suggest that there may be a characteristic reaction with PTAH, but this is
insufficient basis for a hypothesis. Moreover, Hrsel himself pointed out exceptions to his rule, mentioning proteins that contain tryptophane but stain
with the light green of his technique.
The most satisfactory explanation of the differential staining with PTAH
and of its alteration by the various pretreatments which have been employed
in this investigation appears to be that suggested by Baker (1958) for staining
with acid dyes generally. Thus, the red PTAH complex seems to be of larger
molecular size than the blue complex and, correspondingly, the aniline blue of
Mallory's mixture is of larger molecular size than the orange G. Collagen is
permeated by the largest molecules, the red PTAH and the aniline blue, and
muscle by the smaller blue PTAH complex and the orange G. Red cells are
permeated ohly by the orange G, which is presumably of still smaller molecular
size than the blue PTAH complex. The effects of some of the pretreatments
which were employed may be due simply to interference with the configuration of the protein molecule rather than to removal of any specific reacting
group. Alkaline hydrolysis, for instance, appears to 'open up' the muscle
protein sufficiently for the entry of the largest molecules, and the red cell
protein sufficiently for the entry of the intermediate blue PTAH complex.
The effect of methylation may possibly have a similar basis. The actions of
chromium mordanting in preventing the element of fibre stain in muscle and
in restoring normal staining of muscle after methylation or alkaline hydrolysis
may be due to the formation of cross-linkages in the tissue protein, reducing
its permeability. On the other hand, mercurial mordanting has the same
effects on the staining picture, though mercury is said not to form crosslinkages (Pearse, i960). However, the reactions of mercuric chloride with
tissue proteins are far from clear (Baker, 1958).
It might be expected that if the configuration of a protein permits the entry
of a particular dye or dye complex, certain specific groups which will bind the
dye must also be present for staining to occur. The results of the blocking
techniques give no very firm evidence of what groups are involved in the
binding of the different dyes. The failure of nitrosation to have any significant effect on the staining of the thicker collagen fibres suggests the participation there of groups other than primary and secondary amines. The very
gross impairment in the staining of collagen produced by benzoylation indicates 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 responses to blocking techniques. Thus, the staining of fine collagen fibres is
considerably reduced by nitrosation, while Monne and Slautterback (1951)
found that deamination completely abolished aniline-blue staining in the yolk
of sea-urchin eggs. It is possible that amino-groups and other groups are
capable of binding the red PTAH complex or the aniline blue of Mallory's
mixture, but that in fine collagen fibres and in the yolk of sea-urchin eggs the
amino-groups predominate while in thicker collagen fibres other groups are
more important. Alternatively, and much more probably, the basic groups
of the thick collagen fibres may simply be more resistant to nitrosation, either
for physical or chemical reasons. It has been pointed out by Terner and
Clark (i960) that substitution in amino-groups may make them resistant to
nitrosation, though they continue to bind acid dyes.
The hydroxyl groups of hydroxy-proline may possibly be involved in the
binding of the red PTAH complex to collagen, but the elution of the PTAH
by strong urea solution is hardly sufficient evidence to suggest that hydrogen
bonding is concerned in this when mild alkaline hydrolysis is equally effective.
However, the rapid elution of aniline blue by urea solution suggests that
hydrogen bonding may be involved in the binding of aniline blue to collagen.
Gustavson (1957) has considered that the amphionic dye benzopurpurine 4B
is held by hydrogen bonding to the hydroxyl groups of collagen, and a similar
explanation may account for the great affinity of aniline blue for collagen.
Puchtler and Isler (1958) explained the binding of aniline blue to collagen in
the presence of PMA by suggesting that the dye was held by free acidic groups
of tissue-bound complex acid. There is no direct evidence that this is true, or
that the reduction in the staining of collagen by aniline blue when the dye is
used in conjunction with complex acid instead of in simple acid solution is
not due to competition for binding sites between dye and polyacid (Baker,
1958).
The failure of muscle to stain with short exposure to PTAH after acetylation, phosphorylation and deamination implies that amino-groups and
probably other basic groups and hydroxyl groups are involved in the binding
of the blue PTAH complex. The conversion of the blue staining to red by
methylation or alkaline hydrolysis may be due, as has already been suggested,
to rupture of intramolecular bonds in the muscle protein. Obviously methylation, which has been claimed to be specific for carboxyl groups (FraenkelConrat and Olcott, 1945), would be expected to affect linkages between
carboxyl groups and other groups. The very mild and brief alkaline hydrolysis
may break ionic linkages, and the similar though less complete effect of urea
solution suggests that rupture of hydrogen bonds may be involved in the
alteration of the staining reaction of muscle. On the other hand, it is possible
that carboxyl groups are necessary for the binding of the blue complex to
muscle, and this is suggested by the partial restoration of blue staini ng after
permanganate '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 PTAH
complex, it is difficult to explain the increased blue staining after mercurial or
chromium mordanting—which, at any rate according to some authorities
(Pearse, i960)—would be expected to block carboxyl groups. There is, however, an indication that acidic groups may be involved in the binding of
complex acid to nuclei, in that treatment with PMA or PTA greatly reduces
the normal nuclear basiphilia. After methylation, treatment with complex
acid induces a nuclear basiphilia comparable with that occurring in collagen.
The blue and the red staining with PTAH must be due to different polyacid / dye complexes and in some respects the PTAH mixture resembles the
lakes of PTA with basic dyes (Pratt, 1947). Thus an increase in polyacid
content or heating of the mixture lightens the colour. In addition, the similarity of the staining distribution of the red PTAH complex with that of the
basic thiazine dyes after pretreatment of the section with PMA or PTA suggests
that the red PTAH may be a complex of similar structure to the lakes of PTA
with 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 isoelectric point of about 6-5 (Baker, 1958). Possibly the blue complex is of
a similar nature, but with a lower polyacid content and molecular size. On
the 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 course
likely that both red and blue staining are each due to mixtures of several
different complexes.
No complete explanation can be offered for the staining reactions of tissue
tonstituents with the various preparations which have been studied, but it is
clear that differential staining with all of them is dependent upon similar
fundamental principles. The most satisfactory approach appears to be that of
relating the staining by different dye complexes with the permeability of
tissue proteins to molecules of differing sizes, as Baker has described. Tissue
groups must also be available to bind the dye complex once it has permeated
the protein, but until more specific chemical techniques can be applied it is
impossible to define these precisely. In conclusion it is worth emphasizing
that the results of blocking techniques in an investigation of this kind must be
interpreted with caution. Effects on staining reactions may often be due to
interference with the normal configuration of the protein molecule rather
than to blockade of a specific reactive group.
My thanks are due to Professor R. D. Lockhart, in whose Department the
initial part of the work was carried out, and to Professor G. A. G. Mitchell
for his valuable advice on the preparation of this paper.
Buhner—Phosphotungstic acid / haematoxylin
323
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