Ultrastructural cytochemistry of the diaminobenzidine reaction in the aquatic fungus Entophlyctis variabilis

Ultrastructural localization of peroxidatic activity was investigated in the chytrid Entophlyctis variabilis with the 3,3-diaminobenzidine (DAB) cytochemical prodedure. The subcellular distribution of reaction product varied with changes in pH of the DAB medium and with the developmental stage of the fungus. Incubations in the DAB reaction medium at pH 9.2 produced an electron dense reaction product within single membrane bounded organelles which resembled microbodies but which varied in shapes from elongate to oval. At this pH the cell wall also stained darkly. When the pH of the DAB medium was lowered to pH 8.2 or 7.0, DAB oxidation product was localized within mitochondrial cristae as well as in microbodies and zoosporangial walls. As soon as zoospores were completely cleaved out of the zoosporangial cytoplasm, endoplasmic reticulum (ER) also stained. When the wall appeared around the encysted zoospore, ER staining was no longer found. The influence of the catalase inhibitor, aminotriazole, and the inhibitors of heme enzymes, sodium azide and sodium cyanide, on the staining patterns within cells incubated in the DAB media indicates that microbody staining is due to both catalase and peroxidase, mitochondrial staining is due to cytochrome c, and ER staining is due to peroxidase.Abbreviations DAB 3,3-diaminobenzidine-HCl - ER endoplasmic reticulum     

Ultrastructural localization of oxidative and peroxidative activities in a carrot suspension cell culture

Summary Oxidative and peroxidative activities were localized at the ultrastructural level in suspension cells of an anthocyanin-producing strain of carrot after treatment with dihydroxyphenylalanine (DOPA) and diaminobenzidine (DAB). In DOPA-treated cells a reaction ascribed to polyphenoloxidase (PPO) occurred in the thylakoids of plastids. After DAB treatment at pH 9.0 reactions occurred in microbodies and plastid thylakoids; after treatment at pH 6.8 additional reactions occurred in the mitochondrial cristae and cytoplasmic ground substance. No reaction occurred in the cell walls at either pH. A reaction could not be unequivocally detected in the vacuoles because of the natural occurrence of osmiophilic material. Application of peroxidase and PPO inhibitors indicated that four distinct systems were involved in the DAB reactions: catalase was correlated with the reaction in the microbodies, peroxidase with the reaction in the cytoplasmic ground substance, cytochromes with the mitochondrial reaction, and PPO with the reaction in the thylakoids of the plastids.Contribution No. 1964 of the Rhode Island Agricultural Experiment Station.     

The use of diaminobenzidine (DAB) for the histochemical demonstration of cytochrome oxidase activity in unfixed plant tissues

Summary Cytochrome oxidase activity was demonstrated in unfixed root segments from Lupinus albus at the ultrastructural level using the osmiophilic reagent 3,3-diaminobenzidine (DAB). Precipitate, the formation of which was completely inhibited by 0.01 M KCN, and observed almost entirely on mitochondrial cristae, is considered to be produced by cytochrome oxidase activity. Heterogeneity of mitochondria as to the intensity of the reaction in the same cell could not be established with certainity. However, mitochondria of the root tip cells and cells belonging to the plerome consistently did not show histochemically demonstrable cytochrome oxidase activity.     

Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry

Three metallic ions, NiCl2, CoCl2, and CuSO4, were found to modify the color of the normally brown diaminobenzidine (DAB) reaction. The colors ranged from purplish blue (NiCl2), dark blue/bluish black (CoCl2), to greyish blue (CuSO4). We have found that the CoCl2 + DAB is the ion of choice because: 1) it yields a distinct dark blue color that is easily distinguishable from brown DAB; 2) the blue reaction product is very stable throughout the entire staining procedure; and 3) background staining is minimal. These findings can be applied to the double staining technique of two different antigens in the same section. Among three staining procedures discussed, the avidin-biotin peroxidase complex (Co-DAB)-peroxidase-antiperoxidase (DAB) technique produced the best results because: 1) no antibody elution was needed following the avidin-biotin-peroxidase complex procedure when the CoCl2-DAB modification was used; and 2) no background staining occurred.     

Early Detection of Bacterium-induced Basal Resistance in Tobacco Leaves with Diaminobenzidine and Dichlorofluorescein Diacetate

The present study demonstrate that in tobacco leaves the diaminobenzidine (DAB) and 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) staining is a useful indicator of the basal (also known as general or innate) defence‐associated reactions, especially of the early developing form of basal resistance (EBR). DAB and DCFH‐DA, in the presence of H₂O₂ and peroxidase converts to a brown polymer and fluorescent DCF respectively. In the present study, the hypersensitive response (HR)‐inducing avirulent Pseudomonas syringae pv. syringae 61, its HR‐negative hrp/hrc mutants and even non‐pathogenic bacteria such as P. fluorescens and Escherichia coli caused DAB and DCFH‐DA staining, if the dyes were injected 3–4 h after bacterial inoculation into tobacco leaves. The conditions that enable the staining of plant leaves infiltrated with HR‐negative bacteria were persisted for 1 to several days depending on the physiological state of the plant, and plant activity was required to the development of the staining. The live virulent P. syringae pv. tabaci was able to suppress the development of the staining reaction. Bacteria that induced more intensive staining reaction triggered stronger local resistance response, which was verified by its ability to inhibit the HR by challenging avirulent bacteria and by expression analysis of genes that are activated during the basal defence response. The peroxidase enzyme activity increased in bacterially treated tobacco tissue, and inhibition of peroxidase activity blocked the development of the staining. The results showed that in tobacco leaves the staining reactions were associated with the general recognition and basal defence reaction of tobacco plant and can be used as markers in tobacco leaves for testing the occurrence of this type of defence.     

A comparative histochemical study of peroxidase activity in the submandibular glands of five mammalian species including man

Summary A light microscopic histochemical investigation of endogenous peroxidase activity in specimens of the submandibular salivary glands of man, hamster, rabbit, dog and guinea pig was carried out. A modification of the original Graham and Karnovsky diaminobenzidine (DAB)-hydrogen peroxide method was employed at different pH's.At all pH's (6.0, 7.6, and 9.0) a positive DAB reaction was found: in serous acinar cells in four of seven human submandibular glands, in convoluted tubule cells of the hamster, in acinar tissue, in secretory granular tubule cells and in the saliva of the guinea pig. This staining pattern was not markedly affected by KCN or 2,4-dichlorophenol (DCP). Furthermore, small cytoplasmic granules in collecting ducts of the dog displayed positive, KCN- and DCP-resistant DAB staining at all pH's tested. No reaction was observed in the acinar cells of the dog and rabbit glands.Mitochondrial oxidation of DAB in the striated duct cells occurred in all of the glands examined. Optimal staining of these cells was obtained at pH 6.0, but there was also strong positive staining at pH 7.6. At pH 9.0, however, the staining of the striated duct cells was very faint. The positive reaction in the striated duct cells was completely abolished by KCN.     

In situ detection of spontaneous superoxide anion and singlet oxygen production by mitochondria in rat liver and small intestine

In the present study, the endogenous formation of reactive oxygen species was localized in rat liver and small intestine. The 3,3′-diaminobenzidine (DAB)-Mn²⁺ technique in which cobalt ions were included in the incubation medium was applied to unfixed cryostat sections of intact tissues. Addition of manganese ions to the DAB-Co²⁺- containing medium greatly increased the amounts of final reaction product formed compared with incubations with only DAB and cobalt ions. In liver, a blue final reaction product was deposited, particularly in hepatocytes surrounding portal tracts. In the small intestine, the DAB--cobalt complex was mainly found at the basal side of enterocytes. Goblet cells remained unstained. Electron microscopical images revealed that an electron-dense reaction product was exclusively present at both inner and outer membranes and at the intermembrane space in mitochondria of liver parenchymal cells and duodenal enterocytes. It was shown that the spontaneous formation of final reaction product was enzymatic and dependent on the presence of oxygen in the medium. Sulphide decreased the reaction, which may indicate that cytochrome c oxidase was partially involved. Benzoquinone and histidine, which are scavengers of superoxide anions and singlet oxygen respectively, reduced the amount of final reaction product considerably. Furthermore, the formation of final reaction product was sensitive to specific inhibitors of NADH:coenzyme Q reductase and aldehydeoxidase, indicating that these enzymes were at least partly responsible for the generation of superoxide anions and singlet oxygen and for the formation of the DAB--cobalt complex. This revised version was published online in November 2006 with corrections to the Cover Date.     

Endogenous cytochrome c and cytochrome oxidase in rat and mouse kidney: light microscopic histochemical study.

Activity of cytochrome c oxidase and the level of endogenous cytochrome c were investigated light microscopically in adult rat and mouse kidney by incubating unfixed frozen sections with diaminobenzidine (DAB) in the absence or presence of exogenous cytochrome c. The results suggest that DAB staining intensity mainly reflects the local density of mitochondria and only occasionally visualizes the differences in cytochrome oxidase activity and/or endogenous cytochrome c content. Most intense reaction was observed in proximal and distal tubules both in rat and mouse. Finer differentiation of reactivity in particular nephron segments and interspecies differences between rat and mouse kidney are also described.     

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes d-aminoacid oxidase and l--hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.     

An ultrastructural assessment of mitochondria in the gastric parietal cell with the high iron cliamine method

Summary The ultrastructural densification of mitochondria by the high iron diamine (HID) method has been investigated by staining gastric parietal cells with each component of the HID reagent, alone or in combination, or with an otherwise modified HID solution. The effect of certain chemical treatments prior to staining has also been assessed. These tests provided evidence for at east three cytochemically distinguishable constituents in the mitochondrial matrix. In addition, the results from these tests and observations obtained with a newly introduced diaminobenzidine (DAB)-FeCl₃ staining reagent indicated that the probable mode of action of the HID method in imparting density to mitochondrial matrix entails combination between a complex of iron with polymerized or aggregatedm- andp-diamine and some matrix constituent. Assessment of the cytochemical staining in varied fixation condition revealed that the lucent population of mitochondria recently described in HID-stained parietal cells reflects a failure to stain with the method because of inadequate fixation.The possible nature of the HID-reactive substance in the mitochondrial matrix is discussed in the light of these observations and other cytochemical reactivities.     

A dual-immunocytochemical method to localize c-fos protein in specific neurons based on their content of neuropeptides and connectivity

Enhanced expression of the immediate early gene c-fos has been used as a marker of cellular activation in many different neuronal pathways. We wished to determine the neurochemical content and the connectivity of neurons, in which expression of c-fos is induced. For this purpose, a dual-immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chromogen for c-fos protein located in the nucleus, and benzidine dihydrochloride (BDHC) in the presence of sodium nitroprusside to reveal cytoplasmic antigens (neuropeptide or retrograde tracer) in the same section. The blue granular BDHC reaction product in the cytoplasm combined with the homogeneous brown nuclear DAB staining for c-fos protein provides excellent resolution of dual-labelled cells even in tissue sections of 40 m in thickness. The high sensitivity of the avidin-biotin-peroxidase immunocytochemistry and the stability of the reaction products provide an excellent tool for quantitative analysis of stimulated cells within a neurochemically defined cell group. The BDHC/DAB protocol was developed to identify activated cells in three experimental situations. Firstly, to investigate the phenotype of light-activated cells in the suprachiasmatic nucleus of the hypothalamus, c-fos protein DAB staining was carried out together with BDHC staining for peptide histidine isoleucine (PHI) and vasoactive intestinal peptide (VIP). Secondly, to identify activated neurons in female Syrian hamsters at the time of the proestrous luteinizing hormone surge, c-fos protein staining with DAB was carried out in combination with BDHC staining for gonadotrophin-releasing hormone (GnRH). In both these studies, cells which co-localized the peptide and c-fos protein in the nucleus could be identified unequivocally. Thirdly, to analyse projections of c-fos-immunoreactive neurons, the retrograde tracer, cholera toxin subunit B (ChB) was pressure-injected into the piriform cortex of rats, which were thereafter fully kindled in the contralateral amygdala. The tract tracer was stained with BDHC as the chromogen. Due to the advantages of the dual-labelling methodology, the combination of retrograde tracing and c-fos protein histochemistry provides an excellent method for identifying projecting and activated neurons in the same section.     

Actin localization and function in higher plants

Summary Two different cytochemical methods were used to study the localization of uricase (EC 1.7.3.3) and catalase (EC 1.11.1.6) in developing root nodules of soybean (Glycine max) inoculated as seeds withBradyrhizobium japonicum. One of the methods employs DAB (3,3-diaminobenzidine) and detects uricase activity indirectly by coupling it to endogenous catalase activity. The other method utilizes cerium chloride to detect uricase activity directly. These methods were modified to obtain not only a strong staining reaction but also improved ultrastructural preservation. With the indirect DAB method, intense staining indicative of both uricase and catalase activity was obtained in the enlarged peroxisomes of older uninfected cells. Similar staining was observed in enlarging peroxisomes of younger uninfected cells, and in the material of associated sacs whose bounding membranes appear to arise as distensions of the ER. The observations are discussed in relation to the controversial role of the ER in peroxisome biogenesis. Although the small peroxisome-like organelles of infected cells did not give a clearly positive reaction in the indirect DAB method, they reacted positively in the cerium chloride method, and are considered to be peroxisomes.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum     

Use of 3,3′-diaminobenzidine as a biochemical electron donor for studies on terminal cytochrome oxidase activity inAzotobacter vinelandii

Azotobacter vinelandii cells readily oxidize the dye 3,3′-diaminobenzidine (DAB), which has been previously used as an electron donor for studies on the mitochondrial cytochromec oxidase reaction. The DAB oxidase activity inA. vinelandii cells was 10-fold lower than that noted for theN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase reaction, which is commonly used to measure terminal oxidase activity both in bacteria and mitochondria. Analyses of cell-free extracts show that DAB oxidase activity is concentrated almost exclusively in theA. vinelandii membrane fractions, most notably in the “R₃” electron transport particle (ETP). Oxidation studies, which employed both whole cells and the ETP fraction, show DAB oxidase activity to be markedly sensitive to KCN, NaN₃, and NH₂OH. A manometric assay system was developed which readily measured DAB oxidase activity in bacteria. Preliminary studies indicate that ascorbate-DAB oxidation inAzotobacter vinelandii measures terminal cytochrome oxidase activity in a manner similar to the TMPD oxidase reaction.     

A proton-translocating adenosine triphosphatase is associated with the peroxisomal membrane of yeasts

The association of an ATPase with the yeast peroxisomal membrane was established by both biochemical and cytochemical procedures. Peroxisomes were purified from protoplast homogenates of the methanol-grown yeast Hansenula polymorpha by differential and sucrose gradient centrifugation. Biochemical analysis revealed that ATPase activity was associated with the peroxisomal peak fractions which were identified on the basis of alcohol oxidase and catalase activity. The properties of this ATPase closely resembled those of the mitochondrial ATPase of this yeast. The enzyme was Mg²⁺-dependent, had a pH optimum of approximately 8.5 and was sensitive to N,N-dicyclohexylcarbodiimide (DCCD), oligomycin and azide, but not to vanadate. A major difference was the apparent K _m for ATP which was 4–6 mM for the peroxisomal ATPase compared to 0.6–0.9 mM for the mitochondrial enzyme.Cytochemical experiments indicated that the peroxisomal ATPase was associated with the membranes surrounding these organelles. After incubations with CeCl₃ and ATP specific reaction products were localized on the peroxisomal membrane, both when unfixed isolated peroxisomes or formaldehyde-fixed protoplasts were used. This staining was strictly ATP-dependent; in controls performed i) in the absence of substrate, ii) in the presence of glycerol 2-phosphate instead of ATP, or iii) in the presence of DCCD, staining was invariably absent. Similar staining patterns were observed in subcellular fractions and protoplasts of Candida utilis and Trichosporon cutaneum X4, grown in the presence of ethanol/ethylamine or ethylamine, respectively.Abbreviations MES 2-(N-Morpholino)ethanesulfonic acid - DCCD N,N-dicyclohexylcarbodiimide     

A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland

Summary A technique for the cytochemical demonstration of peroxidase activity in unfixed guinea-pig thyroid tissue is described in this paper. The substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) is oxidized by the peroxidase to form an insoluble reaction product. Optimal results were obtained after 20 min incubation at 37° C in reaction medium containing 1.4mm DAB (in 0.1m Tris-HCl) and 0.15mm hydrogen peroxide at pH 8.0. Peroxidase activity was seen in the thyroid follicle cells as a diffuse brown reaction product (which was more dense and granular in erythrocytes). The enzyme activity was quantified using a scanning-integrating microdensitometer, and the effects of two specific peroxidase inhibitors were evaluated. Both 3-amino-1,2,4-triazole and methimazole inhibited peroxidase activity in the follicle cells (enzyme activity was still seen in the erythrocytes), maximal inhibition occurring at 10mm. Stimulation of peroxidase in the thyroid was observedin vivo (1 I.U. TSH administered every 8 h for two days), with the maximal stimulation occurring after 1 day.     

Ultrastructural demonstration of endogenous peroxidatic activity in mammalian arterial wall

Summary Endogenous peroxidatic activity has been demonstrated at the ultrastructural level in large arteries of rabbit and rat using diaminobenzidine. The reaction was positive in endothelial cells of both species and also in the smooth muscle cells of rat arteries. The reaction product was localized in the nuclear envelope and endoplasmic reticulum of the reactive cells. Since the enzymatic activity was extremely sensitive to fixation, best visualization was obtained in unfixed, directly incubated tissues in which additional mitochondrial staining occurred due to the activity of endogenous cytochrome c/cytochrome oxidase system. The peroxidatic activity was partially sensitive to cyanide and could be completely abolished by azide and aminotriazole. It has been suggested that the observed endogenous peroxidatic activity of the arterial wall components reflects the activity of prostaglandin endoperoxide synthetase and, indirectly, production of prostacyclin (PGI₂).Preliminary results were presented at the VIth International Histochemistry and Cytochemistry Congress in Brighton, England, 17–22 August 1980     

Selective cytochemical localization of peroxidase,cytochrome oxidase and catalase in rat liver with 3,3′-diaminobenzidine

Summary In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H₂O₂. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H₂O₂. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

Cytochrome oxidase histochemistry in the rat hippocampus

Summary The diaminobenzidine (DAB) method was adapted for the microphotometric determination of cytochrome c oxidase (cyt ox) in the rat hippocampus. The qualitative and quantitative investigations at the light microscopic level showed that acetone and cytochrome c pretreatment of cryostat sections resulted in a significant increase of demonstrable cyt ox activities. The final incubation medium consisted of 7.5 mM DAB, 2% polyvinylalcohol (PVA) and 6% dimethyl sulfoxide in 0.1 M Hepes buffer; final pH 7.5. PVA was used to keep DAB and artificially oxidized DAB in solution. In the kinetic and endpoint measurements a linear response of the reaction with highest slope was observed only in the initial 5–6 min of reaction. Thereafter the slope decreased. Ultracytochemical demonstrations, which were performed as a topochemical control, showed reaction product only in mitochondria (cristae and intermembranous space). In contrast to vibrotome sections all mitochondria reacted positively in cryostat sections of aldehyde-fixed hippocampi. The enhancement of reaction after acetone pretreatment of cryostat sections (light microscopic level) and after a freezing step in ultracytochemistry is discussed in connection with diffusion problems of DAB through mitochondrial membranes.Dedicated to Professor Dr. G. Lang on the occasion of his 65th birthdaySupported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1)     

Morphological heterogeneity of HeLa cell mitochondria visualized by a modified diaminobenzidine staining technique.

The diaminobenzidine (DAB) technique for the ultrastructural localization of sites of cytochrome c oxidase activity in animal tissues has been adapted to the visualization of mitochondria in animal cells growing in culture. The modified technique allows the staining of mitochondria in all cells in coverslip preparatins for light microscopy. Electron microscopy of thin sections of material treated by this method has revealed that all mitochondrial profiles within a cell (and only these) are stained and they exhibit a well preserved size and internal structure. Coverslip cultures of synchronized and unsynchronized HeLa (F-315) cells stained with the DAB reaction were examined under oil immersion. In the majority of the cells, mitochondria were recognized as discrete bodies in the thinner peripheral portion of the cytoplasm. This observation indicates that in a large proportion of HeLa F-315 cells, at least under the growth conditions used here, the mitochondrial complement is dividied into distinct organelles. This examination also revealed a considerable morphological heterogeneity of mitochondria, which exhibited an ovoid or short rod-like or, less frequently, long filamentous shape, with some evidence of branching. The variability in mitochondrial morphology appeared to be far more prounced between different cells than within individual cells; this cellular heterogeneity was not related in any obvious way to cell-cycle-dependent changes.     

Enzyme histochemical discrimination between tryptase and chymase in mast cells of human gut

We tested four synthetic substances for their histochemical value to demonstrate the catalytic activities of chymase or tryptase in mast cells in sections of human gut. Both Suc-Ala-Ala-Phe-4 methoxy-2-naphthylamide (MNA) and N-acetyl-L-methionine-alpha-naphthyl ester (alpha-N-O-Met) reacted with chymase but not tryptase in mast cells. Conversely, D-Val-Leu-Arg-MNA and Z-Ala-Ala-Lys-MNA were hydrolyzed by mast cell tryptase but not chymase. These results were confirmed by use of two inhibitors of chymotrypsin-like activity, chymostatin and Z-Gly-Leu-Phe-chloromethyl ketone (CK) and two inhibitors of trypsin-like activity, Tos-Lys-CK and D-Val-Leu-Arg-CK. Excellent staining reactions were obtained on cryostat sections of unfixed or aldehyde-fixed tissues and on paraffin sections of Carnoy-fixed tissues. For chymase, however, Suc-Ala-Ala-Phe-MNA is preferred on cryostat sections because it is more specific. On paraffin sections alpha-N-O-Met is preferred because other cells are not then stained. For tryptase, Z-Ala-Ala-Lys-MNA was more selective and more specific and is the preferred general purpose substrate on cryostat sections of aldehyde-fixed tissues and for paraffin sections. D-Val-Leu-Arg-MNA is the preferred substrate for cryostat sections of unfixed tissue. Only a limited number of mast cells showed a reaction for chymase, and these occurred mainly in the submucosa. All mast cells, however, gave a reaction for tryptase, and we recommend the use of either substrate for this enzyme for routine detection of mast cells in human tissues. Double staining for the two main mast cell proteases is most conveniently undertaken on paraffin sections of Carnoy-fixed tissues using MNA substrates for tryptase and alpha-N-O-Met for chymase.