Histochemie 35, 173--188 (1973) �9 by Springer-Verlag 1973

Studies in Succinate Dehydrogenase Histochemistry

Helge Andersen and Poul E. Hoyer

Laboratory of Cyto- and Histochemistry, Anatomy Department A University of Copenhagen, Copenhagen O, Denmark

Received February 2, 1973

Summary. The activity of succinate tetrazolium reductase was investigated in liver and kidney from the rat and mouse. The results obtained were related to the cellular level of suc- cinate dehydrogenase (SDH) as well as to the level of CoQ.

I t was concluded that the low activity in centrolobular areas of the liver lobules compared with the perilobular areas, exclusively is due to a naturally deprivation of Co Q.

The level of SDH as well as of Co Q was very high in renal cortical tubules rich in mito- chondria (proximal and distal convoluted tubules, the ascending thick limb of Henle). This was indicated by the facts that the initial reaction rate was high and no enhancement was obtained by the addition of Co Q10.

In all experiments the activity of fresh frozen sections were compared with the activity of sections from briefly prefixed tissue. The influence of different fixatives, variation in Nitro BT concentration, cryoprotection (dimethyl sulfoxide, DMSO) and osmolar protection (su- crose) was investigated and discussed. Further, the substrate-carrying effect of DMSO was investigated and discussed.

Brief (5 min) fixation at 0 4 ~ C--especially with 1% buffered (pH = 7.2) methanol-free formaldehyde (from paraformaldehyde) gave excellent preservation of morphology and caused no inhibition of SDH activity. Furthermore, the fixation caused an enhancement of Nitro BT penetration into the tissue and an enhancement of formazan substantivity.

The incubation time needed for the appearance of both the red and blue formazan was recorded in order to follow the initial reaction rate. This procedure proved to be a sensitive indicator, when the influence of components added (Co Q10, DMSO, sucrose etc.) was studied.

Introduction

Dehydrogenase his tochemistry has two demands to fulfil: 1) to preserve the quant i t ies of enzymes in a na t ive state inside the tissues and 2) to localize the enzyme ac t iv i ty ma in ta ined in situ.

I n histochemical practice, invest igat ion of succinate dehydrogenase (E.C. 1.3.99.1) ac t iv i ty is commonly carried out by a s t andard succinate te t razol ium reductase technique bu t as stressed very s t rongly by Pearse (1972) the accurate in te rpre ta t ion of the level of succinate te t razol ium reductase ac t iv i ty in a tissue cannot be made wi thout knowledge of the Co Q-level in the mitochondr ia of the cells concerned, since this coenzyme is supposed to connect the f lavoprotein par t of the succinoxidase complex to the cytochrome system and similarely acts as a carrier between the f lavoprotein dehydrogenase and the te t razol ium salt in the histochemical method.

Low ac t iv i ty in cells m a y be due to a low concentra t ion of succinate dehydro- genase (SDH) or to a na tu ra l ly or ar t l factual ly depr ivat ion of mitochondrial Co Q.

174 H. Andersen and P. E. Hoyer:

F ina l ly , i t m a y be due to d e c r e a s e d p e n e t r a t i o n of s u b s t m t e ( succ ina te ) a n d / o r

t c t r a z o l i u m sa l t i n t o d i f f e r en t m i t o e h o n d r i a .

The a im of t h e p r e s e n t s t u d y has b e e n to i n v e s t i g a t e t h e n a m e d d i f f e r en t

f ac to r s d u r i n g t h e h i s t o e h e m i e a l p rocedu re .

Material and Methods

The material comprises 10 (one month old) white female mice (strain CI[) and 10 (one month old) white female rats (Wistar strain) kept in standard conditions and avoiding ex- treme variations in water and salt balance. The animals were sacrificed by cervical dislocation or decapitation. The organs (liver and kidney) to be studied were removed quickly and cut into conveniently thin blocks (1~ 2 mm). From the kidney a specific area was consistently used which included the pyramid with the neighbouring cortex. The liver speciIncns were obtained from the lower half of the left lobe.

Initial Cooling and Sectioning. Unfixed as well as fixed blocks were initially cooled in a C02-expansion cooler with covering device (Pearse-Slee) and then transferred to the cryostat chamber. In instances where several blocks }lad to be used in an experiment, a drop of ice-coht 0.9% NaCl was frozen in the expansion cooler to the block surface to avoid drying during storage.

The specimens were sectioned at 25 ~ in 4 micron sections in a Pearsc-Slee cryostat (tyIJe HI/). The sections were picked up on slides on which a ring-fornmd (diameter -- 17 mm) groove was engraved by aid of an aqueous HF-solution. Ill this way the amount of the in- cubation medium was standardized (5 drops from a Pasteur pipette). To minimize variation in section thickness when different sections have to be compared after incubation, two succeeding sections were always placed within tile ring. The influence of variance in section thickness on the incubation time, needed for initial visual formazan production was further investigated by comparing 4 and 12 micron sections.

In order to avoid drying of the sections (possible inaetivat.ion of enzyme), incubation was performed instantaneously.

Fixation. The preservation of morphoh/gical integrity during initial cooling, sectioning and incubation is an important pre-requisite in order to obtain in situ h)calization of enzyme activities. Accordingly, brief (5 rain) fixation of the tissue blocks at 0 4 ~ C was performed with special reference to stabilization of the different membrane systems of the cells--in particular to prevent loss of Co Q-lecithin complexes from the mitochondria during the histochemical procedure.

Following fixation procedures were performed, using fresh prepared solutions: a) Methanol-free formaldehyde (0.5 1 2 4%) (prepared fronl paraformaldehyde) adjusted

to pH = 7.2 with a 0.2 tool phosphate buffer. In corresponding series suerose (final concentra- tion 0.2 mol) as an osmolar protecting agent and/or dimctbyl sulfoxide (DMSO) (Schuchardt) (final concentration of 5 10%) as a cryoprotective agent were added. Furthermore, fixation with a formaldehyde-calcium solution was performed. The medium was preparcd as a 2% formaldehyde (from paraformaldehyde) (adjusted to pH -- 7.2 with NaOH) to which was added CaCl,_, (anhydrous, gramdar) in a final concentration of 1 mg/ml.

b) Formaldehyde (commercial 40% formalin) as a 1 and 4%, buffered neutral solution in Lillie's modification (Pearse, 1968). As mentioned above sucrose and/or DMSO were added ill parallel series. Furthermore, fixation with formol-calciunl (Lillie, 1965) ill order to form complex coacervatcs of phospholipids with ottler cellular constituents (Pearse, 1968) was used. This fixative was also prepared as 1 and 2 % solutions.

c) As a dialdehyde fixation, glutaraldehyde (1 and 2 %) adjusted to p H - - 7.2 with 0.2 tool phosphate buffer was used. The medium was also used with 11)% DMSO added.

After all fixation procedures the specimens were briefly rinsed in cooled 0.9% NaC1. In all instances sections frmn prefixed tissue were compared with corresponding fresh

frozen sections in order to investigate a possible loss of enzyme activity due to inactivation during fixation.

Cryoprotection. In protecting ('ells against freeze-thaw damage dimethyl sulfoxide (DMSO) has been used. DMSO was partly applied as a constituent of the fixatives as mentioned above

Succinate Dehydrogenase Histochemistry 175

and par t ly used solitary in pre-cooling experiments. In the lat ter cases DMSO was applied as follows: 1) After removal from the animal the tissue was immersed in 10% DMSO (in 0.2 mol phosphate buffer, pH ~ 7.2) for 45 min a t 37 ~ C (Ward and Smith, 1971), 2) same procedure bu t a t 0-4 ~ C and --25 ~ C and 3) same procedure for 5 min a t 0-4 ~ C with and wi thout 0.2 M sucrose added. As a control, tissue was immersed in the buffer for 5 min.

To investigate the role of DMSO (retained in the tissue) as a carrier-agent for the tetra- zolium salt into the tissue, following experiment was carried out: Sections from unfixed and prefixed (1% buffered formaldehyde) tissue as well as from tissue pretreated with 10% buffered DMSO (5 rain a t 0-4 ~ C) were incubated for 15 rain in Nitro BT (0.25 mg/ml) in 0.2 mol phosphate buffer (pH ~ 7.2). Excess of the medium was decanted and the tetrazolium salt was converted to formazan by the reducing agent sodium dithionite (10%) for 10 rain. After a brief rinse in distilled water the sections were embedded in glycerol-gelatine and com- pared under the microscope.

In the same way the possible formazan-extracting effect of DMSO was studied. After the formazan conversion with sodium dithionite, the sections were briefly rinsed in distilled water and placed for 10 min in acetone, DMSO (concentrated) and 10% DMSO respectively. After embedding in glycerol-gelatine the extend of formazan extraction was noticed in the sections by comparing with sections only rinsed briefly in distilled water.

Incubation. As incubation medium the method of Thomas and Pearse (1961) was used as a s tandard procedure al though MTT was replaced by Nitro BT and 0.2 M phosphate buffer was used. In addit ion the concentration of Nitro BT (Sigma) was varied from 0.12 mg/ml to 2.0 mg/ml total incubation medium.

The incubation was carried out in a thermo-regulated water ba th incubator to avoid changes in the incubation medium due to evaporation. Incubat ion temperature was 37 ~ C.

The p i t -va lue in the incubation medium was tested on a pH-meter with a micro electrode uni t (radiometer).

In an a t t empt to increase the penetrat ion of the incubation medium into the sections, a s tandard medium including DMSO (10%) was used in another test series. To control the extract ing effect of 10% DMSO on the two formazans, ordinary incubated sections were t reated with 10% DMSO for 5 min before embedding in glycerol-gelatine.

Beside the s tandard incubation time: 15, 30 and 45 min at 37~ the incubation time needed for the initial, visible (as observed in the light microscope) act ivi ty was in all experi- ments recorded for bo th the red and blue formazans at room temperature (Andersen et al., 1970; Hoyer and Andersen, 1970), since it has been proved tha t the red formazan represent an intermediary reaction product in the enzymatic reduction of the ditetrazolium salt Nitro BT (Eadie et al., 1970; Gabler et al., 1970).

Coenzyme Q. Experiments with eoenzyme Q10 (Sigma) incorporated in the histochemical method were carried out. As coenzyme Q10 remains insoluble in the ordinary incubation medium unless special solvents are included, a coating procedure recommended by Wat ten- berg and Leong (1960) was applied.

"Nothing Dehydrogenase" Reaction. In all the different experiments this reaction was tested by using the conventional incubation medium omitt ing the substrate.

Postincubation Techniques. Preliminary to mounting in glycerol-gelatine, some sections were briefly (1 min) fixed in ice-cold acetone. Others were postfixed in 1% buffered (pH ~ 7.2) formaldehyde (from paraformaldehyde) for 5, 10, 15, 20 and 30 min. These sections were reinvestigated two days later to observe any changes in depositions of formazans.

Results Influence el Type and Concentration el Fixative

Morphological integrity and especially mitochondrial configuration (Fig. 1) was excellently preserved following the brief prefixation of the tissue blocks with 1 % b u f f e r e d f o r m a l d e h y d e ( f rom p a r a f o r m a l d e h y d e ) ( p H ~ 7.2) or 1% b u f f e r e d

n e u t r a l f o r m a l d e h y d e ( f rom c o m m e r c i a l 4 0 % f o r m a l i n ) c o n t a i n i n g 10% D M S O

a n d 0.2 M sucrose . I n t h i s r e s p e c t t h e t w o f i x a t i v e s were s u p e r i o r t o t h e o t h e r

f i x a t i o n p r o c e d u r e s as wel l as t o f r e sh f r o z e n t i s sue .

176 H. Andersen and P. E. Hoyer:

Fig. 1 a d. Succinie dehydrogenase (eoenzyme Q10 incorporated) activity in proximttl tubules from the rat kidney, a, fresh frozen section; b, section from tissue pretreated with I0% DMSO, 0.2tool sucrose and 0.2mol phosphate buffer (pH--7.2); e, section from tissue prefixed in 1% formaldehyde (from 40% commercial formalin), 10% DMSO, 0.2 tool sucrose and 0.2 tool phosphate buffer (pH = 7.2) ; d, section from tissue prefixed in 1% formaldehyde

(prepared from paraformaldehyde) in 0.2 tool phosphate buffer (pH 7.2). X 680

No decrease in SDH ac t iv i ty was noticed following the brief pref ixat ion with

the named two f ixat ives as compared with fresh frozen sections. On the contrary, the incubat ion t ime needed for the appearance of formazans was clearly lowered.

Formol-calc ium f ixat ion was followed by a decrease in the incubat ion t ime needed for init ial visual formazan product ion as compared with fresh frozen sections,

a l though this decrease was not as pronounced as for the above ment ioned two fixatives. This f ixat ion too gave excellent preservat ion of mitochondria. Glutar

Succinate Dehydrogenase Histochemistry 177

aldehyde, especially at high concentrations caused a diffuse (even nuclear) de- position of formazan granules, which was accentuated by storage of the mounted sections. Furthermore, glutaraldehyde (even at low concentrations) caused a pronounced decrease in the overall SDI-I activity as compared with fresh frozen sections.

Influence o/DMSO. When DMSO was used in precooling experiments, mor- phological distortion (shrinkage of cells) was seen after 45 mill application, especially pronounced at 37 ~ C. After the t reatment for 5 min at 0-4 ~ C the pre- servation of the overall morphology was highly improved but was of the same order as seen in fresh frozen sections and particularly, the mitochondrial con- figuration was not better preserved (swelling of mitochondria). When 0.2 tool sucrose was added no improvement of morphology was noticed (Fig. 1 b).

As a constituent of the fixatives, DMSO combined with 1% buffered neutral methanol-containing formaldehyde (from 40 % formalin) (with and without sucrose added) gave an excellent preservation of the overall morphology and especially of mitochondrial configuration, which latter was of the same order as obtained with 1% buffered (pit = 7.2) formaldehyde (from paraformaldehyde) (Fig. 1 c and d). On the other hand no improvement was observed when DMSO was added to 1% buffered formaldehyde (methanol-free) prepared from paraformaldehyde.

In regard to the activity of SDH, the addition of DMSO to the fixatives presumably had no inhibitory effect.

The experiments with formazan production by sodium dithionite revealed: In contrast to the fresh frozen sections, both the sections from prefixed as well as those from DMSO-pretreated tissues showed a stronger formazan deposition. Such depositions were also noticed in the nuclei, although extremely faint compared to the cytoplasmic ones. Post- t reatment of the sections with acetone and DMSO (concentrated) caused a nearly equal and pronounced extractions of formazans, while 10% DMSO only caused a slight extraction. In all instances the most pronounced effect was noticed in fresh frozen sections compared with prefixed sections.

Influence o] Sucrose. The addition of 0.2 M sucrose does not seem to influence the preservation of morphological integrity. On the other hand, it has no in- hibitory effect on the SDIt activity.

Succinate Dehydrogenase Activity in Liver and Kidney. Fresh frozen sections from the rat liver incubated in the standard medium showed an initial red formazan within 3 min and a blue one within l0 min in all liver cells. In sections from liver prefixed in the three fixatives, which were shown to be superior to the others, the red formazan appeared within 2 min of incubation and particularly the blue one within 4 min.

Prolonged incubation of sections from both unfixed and prefixed tissue caused the well known activity pat tern in the liver lobules, the strongest activity now being localized to the perilobular areas.

When DMSO was applied as a constituent of the standard incubation medium, the incubation time needed for the appearance of the formazans was lowered. Especially, this effect was pronounced when dealing with fresh fl'ozen sections in which the red formazan now appeared 1/2-1 rain and particularly the blue one

1 2 a nistochemie, Bd. 35

178 H. Andersen and P. E. Hoyer:

Fig. 2a and b. Suecinate dehydrogenase activity in rat liver lobules, a, coenzyme Q10 not incorporated; b, with coenzyme QJo incorporated. ~ 107

3 4 rain earlier, whereas the shortening in incuba t ion t ime for t i le corresponding sections from pref ixed tissue was negligible. The add i t ion of DMSO has no in- fluence on the different ac t iv i ty pa t te rns peri- and eentr i lobular ly .

W h e n o rd ina ry incuba ted sections were pos t t r ea t ed with 10% DMSO, s(nne ex t rac t ion of for lnazan was noticed, especial ly for the red one. Again the effect was most ev ident when dealing with unf ixed tissue.

The incorpora t ion of coenzyme Q~0 in the his tochemical me thod caused an accelerat ion of the react ion rate, ind ica ted by a reduct ion to about the half of incuba t ion t ime needed for appearance of both the formazans. This appl ied to bot.h unf ixed and pref ixed tissues. Fur the rmore , the incorpora t ion brought about an t, c t iv i ty p a t t e r n differe, nt from tha t observed following prolonged incuba t ion in the s t anda rd med ium: No difference between the per i lobular and ccntr i lobular areas in the l iver lobules could then be observed (Fig. 2).

The var ia t ion in concent ra t ion of Ni t ro BT in the scale 0 . 1 2 ~ 1 . 0 mg/ml in the incuba t ion medium showed a propor t ional increase of react ion ra te ind ica ted by a corresponding lowering of the incuba t ion t ime needed for the appearance of the r u e formazans. In contrast , no p ropor t iona l i ty was observed in the scale 1.0 -~2.0 mg/ml which was ind ica ted by a minimal reduction of the incubat ion

Succinate Dehydrogenase Histochemistry 179

t ime recorded for the appearance of the two formazans. The same results were obtained when coenzyme Q10 was incorporated in the method.

For the kidney only the activities in the cortical tubules rich in mitochondria (proximal and distal convoluted tubules and the ascending thick limb of Henle) were recorded. With the ordinary incubation medium and by using fresh frozen sections, the incubation time needed for appearance of the two formazans was 1-11/2 min for the red one and 11/2-2 min for the blue one. For tissues fixed by the three best fixatives the corresponding incubation times were half a min earlier. Due to the very short incubation time needed, the results were obtained from series of sections and from different animals.

The incorporation of coenzyme Qlo in the method brought about no difference in initial reaction rate. This applied to both unfixed and fixed tissues. After prolonged incubation a slight enhancement of the activity was observed only in the distal convoluted tubules and in the ascending thick limb of Itenle.

No "nothing dehydrogenase" reaction was noticed in the liver and kidney when omitting succinate in the incubation medium. The same was the case when coenzyme Q10 and/or DMSO was incorporated in the method, and the sections were incubated in the medium omitting the succinate.

The special coating technique for coenzyme Q10 caused no artifacts in the sections. Neither were any signs of diffusion of enzyme noticed. In all instances where coenzyme Q10 was applied, activity was only localized to the mitochondria, which could be focused against a stainless background.

A comparison between series of sections of 4 and 12 microns in thickness revealed tha t the difference in incubation time needed for initial visual formazan production in no instances exceeded 30 sec.

Postincubation Techniques. When sections were briefly postfixed in ice-cold acetone nothing was left behind of the red formazan, whereas no extraction of the blue formazan could be detected.

Postfixation in 1% buffered formaldehyde (prepared from paraformaldehyde) even for 30 min did not remove any formazans from the section. Reinvestigation two days later revealed tha t postfixation for 15 min was sufficient to prevent any changes in deposition of formazans.

Discussion

The achievement of an in situ localization in histochemical practice makes it a condition tha t morphological integrity is preserved during the different steps of the methodological procedure. The common use of fresh frozen sections do not settle this problem, since both the quenching techniques and the cutting of sections introduce some thermal damage to the tissue (Silcox et al., 1965; Pearse, 1968). Furthermore, when fresh frozen sections are incubated in aqueous media to demonstrate enzyme activities, parts of the section may go into solution and give rise to loss of nitrogenous material (Altman and Chayen, 1965; Butcher, 1971; Altman, 1972a, b).

To stabilize the tissues, fixation procedures as well as cryoprotection and osmolar protection have to be taken into consideration.

12b Histochemie, Bd. 35

180 H. Andersen and P. E. Hoyer:

In the majori ty of studies in which fixation has been employed the attention was focused on avoiding enzyme diffusion artifacts during the incubation. Con- sequently, most authors have applied the procedure on fresh frozen sections without realizing that it has no effect on damage already occurred during quench- ing and sectioning. Further, it does not prevent diffusion of enzyme from the " o p e n " cells into the fixative. Thus, if fixation has to be performed to maintain morphological integrity, it must be used as a tissue block fixation prior to quench- ing and eryostat sectioning. In the present study this was evident, when sections from prefixed tissue were compared with fresh frozen sections, which agrees with the statement made by Lojda (1965a).

In previous studies different fixatives (formaldehyde, glutaraldehyde, acetone etc.) have been employed with variance in concentration, t ime of fixation and in "vehicles" added (Naehlas et al., 1956 ; Novikoff and Arase, 1958 ; Novikoff et al., 1961; I-Iitzeman, 1963- Walker and Seligman, 1963; Hashimoto et al., 1964; Har- donk, 1965; Johnson, 1965; Kalina and Gahan, 1965; Lojda, 1965b; Conklin, 1966; Fahimi and Karnowsky, 1966; Flitney, 1966; Nissen and Andersen, 1968; Jacobsen, 1969; Makita and Sandborn, 1971). Furthermore, they have been used in relation to different dehydrogenases and to different tissues. Accordingly, no rigorous conclusions concerning fixation can then be drawn.

Meanwhile, fixation is not only a question of maintenance of morphology, but has to be related to the preservation of native enzyme activity (avoidance of in- activation of enzymes during fixation) as debated by Pearse (1968).

In the present study, fixation with 1) 1% buffered (pH = 7.2) formaldehyde (prepared from paraformMdehyde), 2) 1% buffered neutral formaldehyde (metha- nol-containing) with 10% DMSO and 0.2 M sucrose added as well as 3) formol- calcium for 5 min at 0-4 ~ C proved to fulfil the demands mentioned above, when dealing with the enzyme system studied. The superiority of fresh prepared formal- dehyde monomers from paraformaldehyde is presumably due to the avoidance of contaminants as methanol (stabihzer) and formic acid both of whieh exist (for methanol: 10-15 %) in commercial 40 % formalin solution. In fixative 2) the effect of methanol is presumably compensated by DMSO and sucrose since the two components have no influence on morphology when added to methanol-free formaldehyde.

Although briefly performed, the fixation acts as a further improvement in the localization of enzyme activities by increasing the substantivity of the formazans. In the present study and in a previous one (Hayer and Andersen, 1970) the effect is most notable for the red formazan and thus favors localization of enzyme activi- ties in cells with low enzyme concentration. In these cells the enzyme activity is only seen as a red formazan, which has been proved to represent an intermediary reaction product in the enzymatic reduction of the ditetrazolium salt Nitro BT (Eadie et al., 1970; Gabler et al., 1970). In accordance with this fact, it must be supposed tha t an enhaneement of formazan substantivity due to the fixation procedure eliminates or extensively reduces a source of error mentioned by Eadie et al. (1970). The named authors found "that. if the tetrazolium salt in the in- cubation medium is becoming exhausted and there are two areas with equal enzyme activities, but if in one area the tetrazolium reduction intermediate product

Succinate Dehydrogenase Histochemistry 181

is soluble in a tissue component, and in the other it is insoluble, then more di- formazan may appear in the area in which the intermediate is soluble"

On the other hand, the influence of fixation on the substantivity of the tetra- zolium salt proper has to be considered. As stated by Pearse (1972), substantivity of the tetrazolium salt (e.g. to proteins and lipoproteins), although insufficiently defined at present generally is an undesirable quality from a histochemical point of view. Although it is important to distinguish substantivity of the tetrazolium salt from substantivity of the corresponding formazan(s) (Seidler and Kunde, 1969) a scrutiny of previous literature reveals tha t in most instances the problem of substantivity has been dealt with in common.

Seidler and Kunde (1969~ report that fixation could not prevent substantivity of the tetrazolium salts, but made no reference to a possible enhancement due to the fixative. The present study add nothing to the problem of tetrazolium salt substantivity proper, but obviously, fixation facilitates the penetration of the tetrazolium salt as well as of other constituents of the incubation medium into the sections. This effect was noticed in the experiments with non-enzymatic reduction of Nitro BT in the sections and in experiments where DMSO was applied as a constituent of the incubation medium for succinate dehydrogenase activity. In the latter, the substrate-carrying effect of DMSO was negligible in sections from prefixed tissues compared with fresh frozen sections.

Furthermore, the minor enhancement of activity in the sections from prefixed tissue seems to indicate that a possible facilitating effect of DMSO on electron transfer from the thiol groups of dehydrogenases to tetrazolium accepters (Makita and Sandborn, 1971) plays a minor role in histochemical practice.

I t is well-known (Pearse, 1968, 1972) tha t the first type of injury in fresh tissues occurs when mitochondria are deprived of their supply of oxygen. Sensitive mitochondria show an increase of permeability of their membranes, and therefore have to be protected.

As showed in the present study, a protection was obtained by the use of a 5 min fixation of tissue in the named three fixatives prior to quenching and sectioning. Here the mitochondrial configuration was preserved and as far as the activity of succinate dehydrogenase was concerned, there was no decrease in activity when compared with the activity in fresh frozen sections. As mentioned above, the fixation obviously enhanced the penetration of the incubation media into the mitochondria (the role of coenzyme Q10: see below).

I t is difficult to explain the chemical background for the brief formaldehyde prefixation of tissues. Low formaldehyde concentration seems to favour the prevalence of the monohydrate, methylene glycol (Pearse, 1968), which is to be regarded as the reactant whenever aquous formaldehyde solutions are used. However, only a small part of the capacity of formaldehyde to form methylene bridges and addition compounds will be used under the conditions of short t ime fixation (Pearse, 1968; Hopwood, 1969).

Dimethyl sulfoxide (DMSO) has been used as an additive in the field of cryobiology for more than a decade but numerous problems still exist concerning its role in protecting the different cells, freezing rate, inhibition of different en- zymes, mode of action etc. (Farrant, 1965; Bickis et al., 1967; Malinin and Perry, 1967 ; Farrant et al., 1967 ; Melnick, 1968 ; Sch6pf-Ebner et al., 1968; Chagnon et al.,

182 H. Andersen and P. E. Hoyer:

1968 ; Cope, 1968; Rapatz and Luyet, 1968 ; Mazur et al., 1969; Pribor and Novak, 1969; van den Berg and Soliman, 1969; Greene etal . , 1970; Hanker etal . , 1970; Ward and Smith, 1971). Furthermore, its substrate-carrying effect has been studied by using it as a constituent in the histochemical incubation media for different enzyme systems (Gander and Moppert, 1969; Makita and Sandborn, 1971; Reiss, 1971). In the present study the use of DMSO as a cryoprotective medium gave excellent preservation of morphology when applied as a constituent in one of the fixatives [1% buffered, neutral formaldehyde (made from commercial 40~ formalin) with added sucrose], while preeooling experiments with DMSO- buffer-sucrose solutions gave inferior results. That may be due to the fact tha t DMSO causes the precipitation of the component salt of many buffers (Cope, 1968). Furthermore, it was shown in the present study tha t the application of DM SO as a constituent of the incubation medium should be used with caution in histochemical practice since it causes an extraction of the intermediate red forma- zan from the section (particularly from fresh frozen sections) and thereby seriously interfers with the interpretation of the enzyme activity as discussed below.

Sucrose has been widely used in osmolar pretection of cells or isolated cell particulates (Barka and Anderson, 1963; Pease, 1964; Lojda, 1965b; Dariush Fahimi and Karnowsky, 1966; Flitney, 1966; Seligman et al., 1967; Sj6strand, 1967; Hajds and Kerpel-Fronius, 1970; Lis3~ etal . , 1971) in the field of histo- chemistry and electron microscopy as well as iu the isolation of single cells for biochemical analysis of enzyme activities. However, as stated by Lis3~ et al. (1971), little is known about the effect sucrose might have on enzyme activities. The named authors observed that succinate dehydrogenase and lactate dehydrogenase seriously could be inhibited by sucrose if the sucrose was not washed out of the tissue before investigating the enzyme activity.

As shown in the present study, the applied concentration of sucrose in the fixative obviously does not seem to influence the activity of succinate dehydro- genase.

In most histochemical studies only little attention has been paid to the role of and importance of the red intermediary reaction product, which appears in the reduction of the ditetrazolium salt Nitro BT. In general there is no reference to the red formazan at all, and very often it has been removed by acetone or ethanol extraction [Williams and Whiteley, 1963; Andersen, 1965; Johnson, 1965, 1967; Meier-Ruge (electron microscopical study), 1965; McMillan, 1967; Abdulla et al., 1968 ; Jacobsen, 1969; Dahl and Mellgren, 1970; Winkler, 1970] before the sections were investigated for enzyme activities. The red formazan causes special problems in quanti tat ive dehydrogenase histochemistry (Eadie et al., 1970; Gabler et al., 1970; Altman, 1972) since in undehydrated sections there would be a difficulty in measuring two different coloured reduction products. Accordingly, most authors neglect the red formazan, although Eadie et al. (1970) claimed that in quanti tat ive histochemistry it is essential to calibrate reactions involving ditetrazolium re- duction.

As stated in a recent paper (Hoyer and Andersen, 1970) it is important to record the localization of both the red and blue formazan if sites with low enzyme activities should not be missed, and accordingly sections must be investigated before treated with acetone or ethanol.

Succinate Dehydrogenase Histochemistry 183

The exact formula of the red intermediary reaction product has not been established (Eadie et al., 1970; Pearse, 1972) and according to Pearse (1972) it has not yet been shown conclusively if the production of the different formazan eolours under histochemical conditions are due to isomery, intermediates or half- reduction. Meanwhile, a previous s tudy (Hoyer and Andersen, 1970) reveals that factors influencing the formation of the red formazan (extraction during the in- cubation) seriously effect the formation of the blue one indicated by a pronounced retardation in appearance of the latter even in cells where the enzyme activity is high. Accordingly, solvents like acetone or dimethyl sulfoxide in the incubation medium should be avoided or used with caution.

For the latter, its substrate-carrying effect had to be balanced against its formazan-extracting effect.

In all studies dealing with dehydrogenase histochemistry, recording of the appearance of both the red and blue formazan must be highly recommended. This applies to all the different types of cells under investigation. By using such a procedure, the reaction rate can be followed during the incubation as can the influence of components added (e.g. coenzyme Q, solvents like DMSO etc.), which is not always reflected by the use of a fixed standard incubation time.

The role of coenzyme Q as an intermediary carrier between the flavoprotein part of the succinoxidase complex and the cytochrome system has been well established in the histochemical practice (Wattenberg and Leong, 1960; Jones, 1965 ; Horwitz et al., 1967 ; Wolman and Bubis, 1967 ; Pearse, 1972). Nevertheless, relatively little at tention has been focused to this fact and Pearse (1972) strongly stressed that accurate interpretation of the level of SDH activity in a tissue cannot be made without knowledge of the coenzyme Q level in the mitochondria of the cells concerned. Acetone effectively removes the coenzyme Q (possibly coenzym Q-phospholipid complexes) from the mitochondria (Horwitz et al., 1967 ; Wolman and Bubis, 1967) and abolishes the SDt t activity, which can be restored by addition of coenzyme Q-phospholipids. Accordingly, acetone pretreatment of sections in order to remove lipid droplets (and thereby avoiding formazan de- positions at water-lipid interphases) must be omitted.

From the present s tudy it is evident tha t the incorporation of coenzyme Q10 in the lfistochemical method caused a pronounced acceleration of the reaction rate (near]y double of tha t seen when Q10 was omitted) in all rat liver cells in which Q9 usually predominates. Furthermore, the incorporation brought about an equal activity pat tern in perflobular and centrolobular areas following prolonged in- cubation. The latter observations is in disagreement with a recent one (Nolte and Pette, 1972). The named authors found an activity 1.6 times higher in the peri- portal areas of the lobules than in centrolobular areas, which is much like the observation when Q10 is omitted. The disagreement may be due to the fact tha t Nolte and Pette used another strain of rats as well as phenazine methosulfate (PMS) instead of coenzyme Q. Since PMS has several drawbacks in the histo- chemical procedure (for discussion: see Pearse, 1972) the present authors find tha t Co Q has to be preferred in order to avoid a diffusible factor (PMStt) in the method. Furthermore, by using Co Q it is possible to follow the initial reaction rate under the microscope, while the use of PMS makes it a condition tha t the reaction is carried out in darkness. Contrary to the observations made by Her-

184 H. Andersen and P. E. Hoyer:

witz et al. (1967), the present authors observed some enhancement of activity in the distal convoluted tubules and in the ascending thick limb of Henle of the kidney, when coenzyme Q10 was introduced in the reaction. Possibly, the dis- crepancy is due to the fact that Horwitz et al. investigated the renal cortex in general and made no reference to the different cortical tubules. Further, no large Co Q crystals were observed throughout the sections as described by Horwitz et al., when CoQ was used by the coating procedure. This may be due to the use of different trademarks of Co Q since Wolman and Bubis (1967), using the same Co Q (Sigma) as the present authors, neither made no reference to such crystals. Nor could artifactual binding of the coated CoQ to the section (Horwitz st al., 1967) be detected. In the present study, only mitoehondrial activity was noticed and this applies to sections from both mffixed and prefixed tissues.

High levels of Nitro BT should be used with caution, since a rise in concentra- tion from 1 to 2 mg/ml only gave a minimM increase of the reaction rate indicated by a negligible reduction of the incubation time needed for the appearance of the two formazans. This may be due to the toxic effect of the tetrazolium salt.

From the aforementioned it can be concluded that the different aetivit.y pattern of SDH in the peri- and eentrolobular areas of the liver lobules exclusively has to be related to a naturally deprivation of the mitochondrial Co Q-level in the eentrolobular areas. In summary this conclusion is based on the following: 1. In- troduction of CoQ in the method caused identical activity patterns eentro- lobularly and perilobularly, 2. when the incubation time needed for the appearance of the two formazans was recorded, the incubation time was the same for peri- and eentrolobular areas regardless of the addition of Co Q, 3. Co Q addition caused an equally reduction in incubation time needed for the appearance of the two forma- zans perilobularly and eentrolobularly. Only prolonged incubation brought about the different activity pattern when the addition of CoQ was omitted. This seems to indicate a reaction rate limiting role of the Co Q-level centrolobularly. 4. Release of mitoehondrial membraneous material during the different steps of preparation seems unlikely, since heterogeneous activity pattern following incubation in Co Q-free medium was obtained in both unfixed and fixed tissue. Here it is of special interest that fixation with formaldehyde containing CaC12 has been proved to inhibit the release of phospholipids (Roozemond, 1969). The combination of formaldehyde fixation with osmolar protection (sucrose) and eryoproteetion (DMSO) may act in the same way, since DMSO is supposed to protect cellular membranes by reducing the concentration of salt in equilibrium with ice at any temperature. 5. I t is unlikely too that the low activity in eentrolobular cells has to be related to decreased penetration of sueeinate and/or tetrazolium salt into the mitoehondria of these cells. With the best fixatives, the same reduction of incubation time needed for the appearance of the two formazans was obtained both per t and eentrolobularly when compared with fresh frozen sections. This was also the ease when DMSO was used as a substrate-earrier in the incubation medium. 6. I t is unlikely that the low enzyme activity in the eentrolobular areas is due to diffusion of SDH from these areas during preparation procedures. This is based on the fact that SDH seems to be a structural component of the mitoehondrial eristae (Mahler and Cordes, 1969), and obviously does not show diffusion in histo- chemical practice (Kalina and Gahan, 1965; Matthiessen and yon Billow, 1969;

Succinate Dehydrogenase Histochemistry 185

Andersen et al., 1970). Fu r the rmore , in the present s t u d y a diffusion of enzyme dur ing incuba t ion would have been de tec ted by ex t r ami tochondr i a l (even ext ra- cellularly) fo rmazan p roduc t ion in sections where the slides p re l iminare ly were coated wi th Co Q10. F ina l ly , the incorpora t ion of Co Q in the me thod revealed an equal concent ra t ion of S D H in peri- and centrolobn]ar areas.

A compar ison be tween sections f rom the l iver and sections f rom cort ical areas of the k idney reveals t h a t the concent ra t ion of S D H as well as the Co Q-level of cells f rom cort ical tubules rich in mi toehondr ia (proximal and dis ta l convolu ted tubu les and the ascending th ick l imb of Hen]e) is higher t han in l iver cells.

I n order to avo id fur ther ingredien ts in the incuba t ion med ium the presen t au thor s have omi t t ed the meta l -complex ing agent E D T A (e thy lenediamine te t ra - aceta te) . I n the presen t s t u d y the use of f ixed t issue has been p reva len t and according to Dixon and W e b b (1966) dena tu red pro te in is a much be t t e r meta l - complexing agent t h a n most na t ive proteins .

B y the observance of all the named controls we agree in the s t a t e m e n t of Bu tche r (1970) t h a t succinate ox ida t ion can be s tud ied q u a n t i t a t i v e l y in i n t a c t sections wi th the same precision as in more convent ional b iochemical isolates, in which the mi tochondr ia are depr ived of the i r na tu ra l envi ronments .

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Dr. Helge Andersen Dr. Poul E. Hoyer Laboratory of Cyto- and Histoehemistry Anatomy Department A University of Copenhagen Universitetsparken 1 2100 ()openhagen Denmark