THE USE OF TETRANITRO-BLUE TETRAZOLIUM FOR THE CYTOCHEMICAL LOCALIZATION OF SUCCINIC DEHYDROGENASE : Cytochemical and Cytological Studies of Sarcoma 37 Ascites Tumor Cells

Succinic dehydrogenase (SDH) was localized in the mitochondria of Sarcoma 37 ascites tumor cells by the use of tetranitro-BT (TNBT) and nitro-BT (NBT) in smear preparations. Results with each tetrazolium salt as electron acceptor were evaluated with respect to: (a) size and shape of the formazan precipitate relative to standard mitochondrial morphology; (b) crystallization phenomena of reduced dye; (c) lipid adsorption of formazan. The association of formazan- or iron hematoxylin-stained mitochondria with lipid droplets within the cells was investigated, as was also the influence of formalin fixation, with and without cold acetone pretreatment, on mitochondrial morphology and enzymatic staining. Data from these studies appear to indicate that TNBT is more suitable than NBT for use as a cytochemical reagent in oxidative and/or dehydrogenase enzyme histochemistry and cytochemistry.     

Cytochemical properties of mitochondria in the gastric parietal cell.

The matrix of some mitochondria in gastric parietal cells of rat and guinea pig evidenced affinity for the high iron diamine method which localizes sulfated complex carbohydrates selectively by light and electron microscopy. Such staining has not been observed elsewhere in the stomach. The high iron diamine reactive mitochondria about equaled in number those which were unreactive, and the two groups were indistinguishable morphologically. The distinction was not apparent either when mitochondria were stained by other cytochemical procedures including dialyzed iron for acidic complex carbohydrates, 3-3' diaminobenzidine-H2O2 at pH 6.0 for cytochrome oxidase, and Kominick's pyroantimonate osmium tetroxide for antimonate precipitable cations. The dialyzed iron method stained acid glycoconjugates in the outer intermembrane space in parietal cell mitochondria. These mitochondria stained more strongly with dialyzed iron than have any others examined heretofore with this method and comprised the only reactive mitochondria in the stomach. Parietal cell mitochondria also stained strongly for cytochrome oxidase but those of other gastric cells failed to evidence this reactivity.     

ULTRASTRUCTURAL DEMONSTRATION OF CYTOCHROME OXIDASE ACTIVITY BY THE NADI REACTION WITH OSMIOPHILIC REAGENTS

A new method for the subcellular and cytochemical demonstration of cytochrome oxidase has been developed with the introduction of N-benzyl-p-phenylenediamine (BPDA) and the discovery that indoanilines are osmiophilic. These indoanilines produced upon oxidation of BPDA in the presence of naphthols are highly colored compounds that yield electron-opaque coordination polymers of osmium (osmium black) that are amorphous, insoluble in water, and in organic solvents. The best methods for preparing rat tissue were in decreasing order: fixation in formaldehyde solution, fresh tissue slices, and frozen sections of fresh or fixed tissue. Ultrathin sections were counterstained by bridging with the thiocarbohydrazide-osmium tetroxide (T-O) procedure for enhancing underlying membranous structures. Cytochrome oxidase activity was noted primarily in mitochondria and occasionally in sarcotubules of heart, in mitochondria and occasionally in infoldings of the plasma membrane of renal tubular cells, and in mitochondria and, to a great extent, in endoplasmic reticulum of hepatic cells. Cytochrome oxidase activity produced deposits in droplet form, whereas dehydrogenase activity resulted in uniform staining of mitochondrial cristae, as recently demonstrated with an osmiophilic tetrazolium salt. Even more recently we have succeeded in demonstrating cytochrome oxidase activity in nondroplet staining on mitochondrial cristae with an osmiophilic benzidine-type reagent that apparently polymerizes upon oxidation (to be published later).     

Detection of glycosaminoglycans in the Golgi complex of chondrocytes

Elongation and sulfation of glycosaminoglycans are pivotal roles of the Golgi complex during the biosynthesis of proteoglycan monomers. In the present work the spatial relationship between these processes has been investigated by using a combination of immunocytochemical and cytochemical techniques. Chondroitin sulfate and keratan sulfate glycosaminoglycans were immunocytochemically localized in 1 to 2 transmost cisternae, also in a system of narrow tubules at the trans face of the Golgi complex of chick epiphyseal chondrocytes. At these same locations sulfate groups were revealed with the high iron diamine (HID) method, proteoglycan monomers being visualized with ruthenium red. Several treatments were assayed in order to reversibly block the secretory pathway. Chondrocytes incubated at a low temperature, 15 degrees C, before fixation, showed both glycosaminoglycans in the middle cisternae of the Golgi stack as well as the above mentioned locations. After low temperature treatment both HID and ruthenium red stained the middle, but not the cis cisternae. Incubation of the cells for 30 min with either diethylcarbamazine or monensin before fixation permitted detection of glycosaminoglycans and proteoglycan monomers in the middle cisternae, whereas HID staining of the Golgi complex, but not that of secretory vesicles, was abolished. The results show that elongation of both chondroitin sulfate and keratan sulfate glycosaminoglycans takes place in the same Golgi compartments. These include the middle cisternae and probably also the trans cisternae and tubules. Also suggested is that sulfation of one or both types of glycosaminoglycans begins in the middle cisternae.     

Subcellular distribution of ionic components in gastric mucosa of the guinea pig

Summary The topographical distribution of cations, anions and polyanions in the guinea-pig stomach has been studied by ultrastructural cytochemical methods. After fixation with the pyroantimonate-osmium tetroxide solution, variable-sized precipitates were localized in the basolateral extracellular space bordering parietal cells or chief cells but not in that bordering mucus-secreting cells. The basal lamina of all gastric cells disclosed a continuous layer of heavy antimonate deposits. Parietal cells disclosed uniformly fine deposits also on the apical plasmalemma both at the main lumen and in the intracellular canaliculi, and revealed, as well, coarse precipitates in the mitochondria. Fixation with a silver acetate-osmium tetroxide solution yielded nitric acid-resistant, silver deposits confined to the luminal surface of the apical plasmalemma in the main lumen and intracellular canaliculi, the lateral intercellular space, the outer surface of the basal plasmalemma and the basal lamina of the parietal cell.Staining with dialyzed iron demonstrated a glycocalyx rich in acid mucosubstance on the basolateral plasmalemma but not on the apical plasmalemma of parietal cells. In contrast, acid glycoconjugate was visualized on the apical plasmalemma of isthmus cells, mucous neck cells and the transitional cell between isthmus and mucous neck cells but little or no acidic glycoconjugate was demonstrated on the basolateral plasmalemma of these cells. The entire plasmalemma of gastroendocrine cells, unlike other epithelial cells, stained uniformly for acidic glycoconjugate. The dialyzed iron and high iron diamine methods stained the outer compartment of mitochondria in parietal cells intensely and that in other gastric cells lightly. These reagents stained the basal lamina of all gastric cells as did ruthenium red. The several characteristic cytochemical properties of parietal cells presumably relate to the unique secretory activity of these cells and are consistent with the view of the intracellular canaliculi of the parietal cell as the main route for hydrogen and chloride ion secretion.     

Localization of ATPase in the endoplasmic reticulum and golgi apparatus of barley aleurone

Summary The cytochemical localization of adenosine triphosphatase (ATPase) was studied in the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya). Isolated barley aleurone layers secrete numerous enzymes having acid phosphatase activity, including ATPase. The secretion of these enzymes was stimulated by incubation of the aleurone layer in gibberellic acid (GA₃). ATPase was localized using the metal-salt method in tissue incubated in CaCl₂ with and without GA₃. In sections of tissue incubated without GA₃, cytochemical staining was confined to a narrow band of cytoplasm adjacent to the starchy endosperm and to the cell wall of the innermost tier of aleurone cells. Cytochemical staining was absent from the organelles of tissues not treated with GA₃. In tissue incubated in the presence of GA₃, cytochemical staining was evident throughout the cytoplasm and cell walls of the tissue. In the cell wall, electron-dense deposits were found only in digested channels. The cell-wall matrix of GA₃-treated aleurone did not stain, indicating that it does not permit diffusion of enzyme. In the cytoplasm of GA₃-treated aleurone, all organelles except microbodies, plastids, and spherosomes stained for ATPase activity; endoplasmic reticulum (ER), Golgi apparatus, and mitochondria showed intense deposits of stain. The ER of the aleurone is a complex system made up of flattened sheets of membrane, which may be associated with both the Golgi apparatus and the plasma membrane. The dictyosome did not stain uniformly for ATPase activity; rather there was a gradation in staining of the cisternae from thecis (lightly stained) to thetrans (heavily stained) face. Vesicles associated with dictyosome cisternae also stained intensely as did the protein bodies of GA₃-treated aleurone cells.     

OSMIUM STAINING OF ENDOPLASMIC RETICULUM AND MITOCHONDRIA IN THE RAT ADRENAL CORTEX

The zona fasciculata of the rat adrenal cortex synthesizes and secretes glucocorticoids. As observed after aldehyde fixation, the cells in this zone contain an extensive endoplasmic reticulum (ER), a small Golgi apparatus, a moderate number of lipid droplets, and abundant mitochondria with tubulovesicular cristae. Numerous areas within the endoplasmic reticulum and mitochondrial cristae appear clear. In addition, a small percentage of mitochondria encompasses large, clear areas. After immersion of finely minced adrenal cortex in unbuffered 2% OsO₄ (40–48 hr at 40°C), deposits of osmium are seen within the Golgi apparatus, the entirety of the ER, and occasionally within mitochondria. In some mitochondria, the deposits are within cristae; in others, within vacuoles; in still others, in both cristae and vacuoles. These localizations correspond best to the clear areas found in aldehyde-fixed tissue. Osmium is not deposited in lipid droplets, in bar-containing inclusions, in mitochondrial matrix inclusions, or in the peripheral, outer mitochondrial spaces. Addition of zinc-iodide to OsO₄ increases the amount of Golgi apparatus and mitochondrial staining. Adrenocorticotropin (ACTH) does not affect the localization of deposits; hypophysectomy decreases mitochondrial staining. This study (a) emphasizes the necessity for electron microscopic confirmation of osmium localization when this technique is used as a Golgi apparatus stain; and (b) suggests that the ER-staining pattern may be consistent in cells actively synthesizing steroids or steroid-like compounds.     

Application of rapid freezing followed by freeze-substitution acrolein fixation for cytochemical studies of the rat stomach

Summary A method involving rapid freezing followed by substitution fixation was developed, using acrolein as a fixative. This was then applied to several cytochemical stainings, and showed well preserved and clear cell structures. Membranes were apparently negatively stained and the ultrastructure of mitochondria, rough endoplasmic reticulum and Golgi apparatus was clearly discernible. The mitochondrial and cytoplasmic matrices were stained rather densely compared with routine chemically fixed preparations, implying a good preservation of matrix substrances. Periodic acid-thiocarbohydrazide-silver proteinate staining was applied to the present method. The mucous granules of surface covering epithelial cells indicated fine staining of bipartite structure and the Golgi apparatus of mucous cells showed clear staining differences based on polarity. Postembedding lectin-ferritin and immunocytochemical stainings were applicable to the present preparations and stable stainings of secretory granules were obtained. A low temperature embedding material, Lowicryl K4M, was also examined. The cell preservation of these samples was not as good as those embedded in Epon, but the rough endoplasmic reticulum and Golgi apparatus of chief cells were stained with anti-pepsinogen antibody as were the secretory granules. The present method was also applicable to light microscopy.     

A permeability test for the study of mitochondrial injury in in vitro cultured heart muscle and endothelioid cells.

A cytochemical permeability test for the detection of injury to in situ mitochondria of cultured heart cells is presented. The test is based on the increased rate at which injured mitochondria stain for succinate dehydrogenase activity. Whereas an intact inner mitochondrial membrane limits the rate at which Nitro Blue tetrazolium and phenazine methosulphate reach succinate dehydrogenase, injured mitochondria allow these reactants to reach the enzyme more rapidly to form microscopically-observable formazan granules. The extent of staining at fixed durations of incubation with the reactants was assessed on a blind basis with pseudo dark-field microscopy, using a standardized rating scale. Differences in the staining of control and treated cells were analysed statistically by a semi-quantitative method. Treatment of the cultures with either vitamin A or chlorpromazine, resulted in more rapid mitochondrial staining. Brief pre-fixation of the cells with cold acetone also labilized the mitochondria as did a delay in the change of culture medium.     

Zur Frage einer Neubildung von Mitochondrien aus Plastiden

Summary In the cells of the foot of young sporophytes ofPolytrichum commune, plastids form buds which may separate entirely from the mother plastid. In ultra-thin sections these bodies may be easily mistaken for mitochondria. However, with the silver staining method ofMarinozzi, the matrix of these bodies has the same density as the matrix of the plastids and is markedly less dense than the matrix of the mitochondria. Similarly, after silver staining the envelope of these bodies resembles the plastid envelope and is distinctly different from the mitochondrial envelope. Thus, there is no evidence that mitochondria originate from plastids, as some authors believe.     

Observations of mitochondria and mitochondrial nuclei by double staining with rhodamine-123 and DAPI in synchronous cultures ofCatharanthus roseus

Mitochondria in cells ofCatharanthus roseus (L.) G. Don in synchronous cell division cultures were observed by double staining using fluorescence microscopy. The cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) first and subsequently stained with rhodamine 123 (r-123). Immediately after staining with r-123, yellowishgreen, elongated and moving mitochondria were observed upon excitation at 485 nm. When the excitation filters were replaced by a UV filter (360 nm), 1 to 7 mitochondrial nucleoids were visible in each mitochondrion in the same field. Changes in the lengths of mitochondria during the cell cycle obtained from the observations under fluorescence microscopy by this staining method suggest the occurrence of multiplication of mitochondria concurrent with the cell cycle ofC. roseus.     

Ultrastructure of proteoglycans in the specific granules of guinea-pig basophilic leukocytes as demonstrated by cuprolinic blue staining

The ultrastructure of sulphate proteoglycans in basophil granules was examined using cytochemical procedures designed to stabilize and visualize these highly anionic macromolecules in situ. Unfixed or glutaraldehyde-prefixed guinea-pig spleen cells were submitted to fixation/staining in 2.5% glutaraldehyde, 0.2% cuprolinic blue (CB; a cationic phthalocyanin dye) and 0.2 or 0.3M MgCl₂ with or without glycosidase treatments. Abundant electron-dense precipitates were present throughout the granule matrix. The stained structures were often arranged in a quasi-crystalline typical banded pattern. Negative control basophils had no electrondense precipitates. Digestion with chondroitinase ABC destroyed the CB-positive electron-dense banded or filamentous patterns while sialidase treatment did not, but led to larger CB-positive filaments in the cytoplasm near the granules. Taking into account their high anionicity, as shown by the stability of dye binding in the presence of 0.3M MgCl₂, and their susceptibility to chondroitinase ABC, the CB-precipitates are assumed to be related to the sulphated proteoglycans previously characterized in basophil granules. The CB-positive crystalline or filamentous network of the granule matrix is also assumed to reflect the in situ location and organization of these intracellular proteoglycans and may be involved in maintaining the shape of the granule.     

A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA

We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient ρ⁻ or ρ⁰ mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methanesulfonate or ultraviolet light. The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter. When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia. Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron. We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells. Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair. These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. Received: 26 March 1999 / Accepted: 29 June 1999     

Microbodies und Diaminobenzidin-Reaktion in den Acetat-Flagellaten Polytomella caeca und Chlorogonium elongatum

Summary In two forms of acetate flagellates, the colourless Volvocale Polytomella caeca and the green Volvocale Chlorogonium elongatum, cell organelles can be demonstrated which are ultrastructurally similar to microbodies of higher organisms. The organelles do not have a close association with the endoplasmic reticulum and are located in the peripheral cytoplasm between the elongated mitochondria. In Polytomella they exhibit more or less spherical profiles in section and have a maximum diameter of approximately 0.2–0.25 . In Chlorogonium the organelles occasionally have an elongated shape and are larger than in Polytomella. Employing the electron microscopic cytochemical reagent diaminobenzidine (DAB)/H₂O₂ to localize the microbodial marker enzyme catalase in these organelles, it was found that no accumulation of the electron-opaque product occurs in the microbodies either at alkaline or neutral pH or at room temperature or 37° C. Only the cristae of mitochondria are stained with the DAB reaction caused by cytochrome oxidase and possibly by a cytochrome peroxidase.Organelles of Polytomella caeca containing catalase or cytochrome oxidase can be separated by rate centrifugation of a crude particulate fraction on a sucrose gradient (Gerhardt, 1971). The particles isolated from the peak of catalase activity show the same fine structural characteristics as the microbodies in situ do. But again, there is no detectable staining of these organelles by the DAB/H₂O₂ reaction.The identity of the microbody-like particles in Polytomella caeca and Chlorogonium elongatum with microbodies in general is deduced despite the negative results in cytochemical localization of catalase in these organelles.     

Comparative study of procedures for histological detection of lectin binding by use of Griffonia simplicifolia agglutinin I and gastrointestinal mucosa of the rat

Summary The histological localisation of -D-galactopyranosyl residues in glycoconjugates of rat stomach and duodenal mucosae was studied by use of Griffonia simplicifolia agglutinin I, i.e. the isolectin mixture (A+B) and the isolectin B₄ (B₄). Cryostat sections which were either unfixed or acetone fixed and paraffin sections from both ethanolacetic acid and formaldehyde fixed tissue blocks were compared. Cellular details were better preserved in paraffin than in cryostat sections. Reactivity of cells binding GS I was less sensitive after formaldehyde than after ethanol-acetic acid fixation inasmuch as higher concentrations of lectins were needed. This drawback could be overcome by trypsinisation of the sections. The binding pattern of GS I (A+B) corresponded with that of GS I (B₄) in either cryostat or paraffin sections. GS I was detected in the cytoplasm of parietal cells and in Brunner's gland cells. In duodenal crypts and villi, lectin was bound to supranuclear regions in the cytoplasm of columnar and goblet cells. The staining efficiency of fluorescein (FITC), horseradish peroxidase (HRP) and colloidal gold particle (CGP) labels in both direct and indirect lectin stainings was compared. Under all experimental conditions, indirect methods required lower concentrations of lectins than direct ones; indirect procedures increased sensitivity about 5–10 fold. CGP labels were always of highest sensitivity when gold particles were further developed by a silver precipitation method. HRP was not as efficient in lectin localisation as CGP, but cytochemical staining was more convenient in routine work. Direct FITC labellings proved to be of lowest sensitivity.     

Ultrastructural visualization of sulphated complex carbohydrates in blood and epithelial cells with the high iron diamine procedure

Synopsis The high iron diamine (HID) method has been found to impart density at the ultrastructural level selectively to sites known to contain sulphated complex carbohydrates. Thus, immature primary granules in rabbit heterophils, immature précrystalloid granules in rabbit eosinophils, all granules of rabbit basophils, mouse and rat mast cells and the nucleoids of -granules of rabbit platelets were stained by HID. Granules of mast cells in rat cervical lymph node varied in the distribution pattern of the HID-reactive component. Mucous droplets within goblets of mouse colonic epithelial cells varied in HID reactivity. Sites known to contain sialomucin but no sulphates, such as mucous cells and apical plasmalemmae in mouse rectosigmoid colon, failed to stain with HID in contrast to their reactivity for dialysed iron at the ultrastructural level. The surface of mast cells and blood cells lacked affinity for HID, indicating that the dialysed iron binding at the surfaces can be attributed to neuraminic acid. HID proved more effective than dialysed iron in visualizing acid mucosubstance in precursor forms of the crystalloid granules in the eosinophil and in mast cell granules. Inclusion of 0.5% glycerol in the HID solution enhanced staining in mouse colon.     

Stereologic studies on mitochondrial configuration in different organs of the rat

Summary Mitochondria from different organs of the rat with configurations ultrastructurally resembling those of isolated mitochondria of known respiratory states have been subjected to Stereologic analysis. Mitochondria were examined from mossy fibers of the granular layer of the cerebellar cortex (condensed state), of the pericentral hepatocytes (orthodox state), and of heart muscle and parietal cells of the gastric fundus (transitional state). In order to study the relationship between mitochondrial compartments and the internal membrane a partition coefficient was introduced, which expresses the volume of the matrix (E_mm) and external compartment (E_ocm) respectively per unit surface area of internal mitochondrial membrane. The Stereologic parameters investigated, i.e. surface density of the mitochondrial membranes, volume density of the mitochondrial compartments and membranes, and partition coefficients generally agreed with the visual evaluation of mitochondrial ultrastructure. However, analysis of the coefficient of variation /x × 100% for E_ocm and E_mm has shown significantly greater variability in the mitochondria of the myocardium than in the gastric mitochondria, despite similar ultrastructure. It is suggested that Stereologic methods, like time-lapse cinematography, give a compound picture of configurational variation and of its plasticity.     

Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase

Summary This paper deals with the progress made over the last few years in our understanding of enzyme cytochemical staining methods as studied using a fundamental approach with the aid of a model system of thin gel films. Although model films with a matrix of polyacrylamide have been mostly used, the properties and possible applications of other matrices are also reviewed. The chemical aspects of the entrapment of enzyme molecules into a matrix are summarized. Special attention has been paid in model film studies to the principles of the trapping reaction of a diffusable precursor resulting from the enzymatic conversion of a substrate. They are considered here as they concern the cytochemical demonstration of acid phosphatase activity with a lead salt. The effect of fixatives on different enzyme activities, the diffusion rate of substrates and chromogenic compounds to the enzyme site, and enzyme kinetics under cytochemical conditions are also discussed, since they are factors which influence the final results of the staining procedures. The advantage of model film studies in enabling the direct correlation of cytochemical and biochemical results is outlined with special reference to the cytochemical determination of glucose-6-phosphate dehydrogenase with Tetra Nitro BT. A method for determining enzyme activities in the soluble fraction of isolated cells after incorporation in model films is described for the first time. This method has proved to be highly appropriate for microscopical observations of glucose-6-phosphate dehydrogenase activity in single cells, because it results in a good morphology and no formazan precipitaties outside the cells. On the other hand, this type of model film forms a bridge between fundamental model film studies using purified enzyme and quantitative enzyme cytochemistry performedin situ.     

Molecular approaches of a mitochondrial mutation inCandida utilis

Analysis of the cytochrome spectra of a mitochondrial mutant ofCandida utilis showed complete absence of apocytochromeb; this suggests a certain degree of damage, probably a small deletion in themit genes of mitochondrial DNA. Oxygen uptake measurements with and without cyanide of the respiratory-competentCandida utilis parent strain and its derivative mitochondrial mutant P_1,2 indicated the absence of the cyanide-sensitive or normal respiratory chain and a lowered rate of cyanide-insensitive or alternate respiration. Mitochondrial profiles and distribution of parental and mutant cells account for an altered mitochondrial DNA which affects mitochondria in the latter cell shape and function. The mutant cells ofCandida utilis were considered asmit ^– mutants from the observations reported here.     

Post-embedding staining with high-iron diamine-thiocarbohydrazide-silver proteinate and its application to visualizing sulfated glycoconjugates in cryofixed kidney and cartilage.

Cryofixation is generally believed to provide optimal tissue preservation. However, certain post-embedding cytochemical reactions, such as high-iron diamine (HID) staining for sulfated glycoconjugates, are not applicable to cryofixed and freeze-substituted tissues. In the present study, the HID technique was therefore adapted for post-embedding staining. HID staining was performed on thin sections of chemically and cryofixed kidney and growth plate cartilage, embedded in Epon and various acrylic-based resins. All resins and most tissue preparation conditions allowed post-embedding staining with HID, albeit to variable degrees. However, no significant cytochemical reaction was obtained with tissue sections of osmicated kidney embedded in Epon. Profile views of re-embedded sections showed that large stain deposits were usually restricted to the surface, whereas small ones were observed throughout the entire thickness of the section. The staining pattern was essentially similar between chemically fixed and cryofixed specimens. In the glomerulus, stain deposits were mainly seen over the free surface of podocyte foot processes and over the lamina rara externa. The pericellular cartilage matrix of chemically fixed specimens often appeared as condensed elements, usually stained with large deposits. In cryofixed tissues this matrix formed a meshwork composed of thin, extended filamentous structures, many of which showed linear arrays of smaller stain deposits. The data presented here indicate that post-embedding HID-TCH-SP staining can be successfully performed on thin sections of tissues embedded in various resins and, as a result, can be further adapted to cryo-prepared specimens to give a high resolution localization of sulfated glycoconjugates in tissues with optimal molecular preservation.