Ultrastructural Demonstration of Cytochrome Oxidase Activity by the Nadi Reaction With Osmiophilic Reagents

Abstract

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 electronopaque 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).In order to develop new cytochemical methods for electron microscopy that would supplement the metal salt methods for hydrolytic enzymes and possibly avoid some of their pitfalls, we have introduced new histochemical principles (5,7,8,20). One principle is based upon the use of organic reactions that yield insoluble pigments which possess an osmiophilic group. The immediate conversion of the reaction product into osmium black on exposure to osmium tetroxide helps to diminish the artifacts due to possible diffusion of the organic reaction product or due to special affinities of reaction product such as lipid solubility. The osmium black so produced is a coordination polymer of osmium (6) and has ideal properties for both light and electron microscopy.In order to do the same for cytochrome oxidase, we could have introduced the osmiophilic group into either of the two components of the Nadi reaction. In our earlier experiments (7,8,20),

An examination of murine foreign body giant cells using cytochemical techniques and thin layer chromatography

“…Cytochemistry : The following cytochemical tests were performed : acid phosphatase ( Barka and Anderson, 1962); alkaline phosphatase (Burstone, 1958); glucose-6-phosphatase (Wachstein and Meisel, 1956) ; glutamate-oxaloacetate transaminase (Papadimitriou and van Duijn, 1970); /%glucuronidase (Fishman and Goldman, 1965); lactate dehydrogenase (Hess, Scarpelli and Pearse, 1958); 5'-nucleotidase (Naidoo and Pratt, 1954); succinate dehydrogenase (Nachlas et al, 1957); oil-red-0 technique for triglyceride (Lille and Ashburn, 1943) ; sodium-potassium-dependent adenosine triphosphatase (Ernst, 1972) ; adenosine triphosphatase (Padykula and Herman, 1955) ; thiamine pyrophosphatase (Novikoff and Goldfischer, 1961); leucine amino-peptidase (Monis, Wasserkrug and Seligman, 1965); peroxidase (Graham and Karnovsky, 1966) ; glucose-6-phosphate dehydrogenase (Pearse, 1972); cytochrome oxidase (Seligman et al, 1967); non-specific esterase (Schnitka and Seligman, 1961); periodic acid Schiff reaction for glycogen and mucopolysaccharides (Pearse, 1968); a-glycerophosphate dehydrogenase (Pearse, 1972); iron (Pearse, 1972); Sudan black for phospholipids (Pearse, 1968) ; toluidine blue (Pearse, 1968); RNA using the methyl pyronin method (Pearse, 1968); glucose-6-phosphatase (Pearse, 1972); isocitrate dehydrogenase (Pearse, 1972) ; malate dehydrogenase (Pearse, 1972) ; NADH dehydrogenase (Pearse, 1972) ; NADP H dehydrogenase (Pearse, 1972); 3 hydroxy-butyrate dehydrogenase (Pearse, 1972).…”

Effect of freezing on cytochrome c oxidase cytochemistry in cells in monolayer culture.

“…This cell line expresses a normal level of COX activity, as measured by the spectrophotometric method (15). The cells were cultured in a Ham Fl 2 medium (Nissui pharmaceutical Co., Ltd., Japan) containing 10% fetal bovine serum, penicillin, streptomycin, and glutamine at 37°C in an atmosphere of 5% CO2 and 95% air, as previously described (14). Enzyme histochemistry by electron microscopy Cells were cultured in polystyrene dishes in a semiconfluent concentration and fixed by being exposed to 2% glutaraldehyde in a 0.1 M cacodylate buffer (pH 7.4) at 4°C for 10 min or 30 min.…”

“…The ultrastructural cytochemical localization of COX activity in mitochondria was first attempted in animal tissues in 1967 (14). The basic method now in use was established by Seligman and his coworkers in 1968 (13), and since then, many reports of electron microscopy studies of COX have appeared (1,2,5,8,11,15).…”

Ultrastructural cytochemical identification of peroxisomes as organelles of terminal oxidation in amitochondrial trichomonad flagellates

“…coworkers used an osmiophilic Nadi reagent (Seligman et al, 1967) to compare the locations of cytochrome oxidase in Plasmodium berghi and 2" gallinaceum; only the latter species has classical mitochondria in the intraerythrocytic trophozoite stage. 2-berghi does, however, dis play classical mitochondria in other life stages (Howells, 1970;Rud^inska and Trager, 1959), (Theakston et al, 1969).…”

“…Nadi method for localization of cytochrome oxidase One other Nadi method, utilizing n-benzyl-p-phenylenediamine and 1-napthol, has been used for ultrastructural cytochemistry with formaldehyde-fixed and fresh rat heart, kidney, and liver (Seligman et al, 1967) and in the amitochondrial phase of, Plasmodium berghi (Theakston et al, 1969) for the localization of cytochrome oxidase. Incubation of rat tissue at pH 7.4 with no substrate resulted in droplet deposition; inherently inferior for enzyme localization with electron microscopy, in mitochondria of heart and kidney, but the liver also exhibited cytoplasmic droplets which were associated either with ER or with microbodies (some microbodies were not reactive).…”