The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylateor phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 ~). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxlde) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that--notable in the case of glutaraldehyde--was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succlnic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.In this paper seven different organic substances are introduced as fixatives for electron microscopy and cytochemistry. The work, as a means of satisfying the requirements of each, has taken into consideration the difficulties experienced when combining the two fields (1, 2). At the present time, osmium tetroxide (3) and, to a lesser extent, potassium permanganate (4) are the fixatives of choice that are currently used in morphological studies with the electron microscope. These reagents provide excellent cytological fixation, and also add density to some of the reactive components of the cells. However, their use in cytochemistry is very limited since they are heavy metal-containing, oxidizing reagents that completely destroy enzymatic activities except after very short periods of fixation (5-9).

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Capillaries were isolated from epididymal fat, and a catecholamine-sensitive adenylate cyclase found in these capillaries was characterized. The effect of various hormones on the accumulation of adenosine 3': 5'-cyclic monophosphate in capillary endothelial cells was determined and the cyclase was found to exhibit mixed alpha and beta characteristics. Cyclase was cytochemically localized in these endothelial cells with 5'-adenylyl-imidodiphosphate as a specific cyclase substrate and alloxan as a specific cyclase inhibitor. Lead imidodiphosphate was precipitated at or near the site of cyclase activity upon hydrolysis of 5'-adenylyl-imidodiphosphate by cyclase. This reaction product was observed primarily on the luminal surface of intact capillaries, in micropinocytic invaginations, in free vesicles within the cytoplasm, and in the intracellular junctions.Digestion of epididymal fat with collagenase has been used by several investigators to obtain pure fractions of fat cells (1, 2). Upon centrifugation of the dissociated adipose tissue, adipocytes collect at the top and the vascular and stromal cells are pelleted. This study describes a method for partial purification of the vascular-stromal pellet to obtain a relatively homogeneous preparation of intact capillaries and reports the biochemical characterization and cytochemical localization of a catecholamine-sensitive adenylate cyclase (cyclase) associated with capillary endothelial cells.A modification of the method of Reik et al. (3) was used in the cytochemical localization of capillary cyclase activity. The substrate, 5'-adenylyl-imidodiphosphate, which is specifically hydrolyzed by cyclase but not by other ATPases (4, 5), was used in comparison with ATP; the specificity of localization was enhanced by a reaction in which lead imidodiphosphate is precipitated at or near the site of cyclase activity. Further specificity was achieved by the use of alloxan, which has been shown to selectively inhibit cyclase while sparing other membrane ATPase (6), and thus would be expected to inhibit product formation when 5'-adenylyl-imidodiphosphate is used as substrate. METHODS AND MATERIALSIsolation of Capillaries. The distal portion of epididymal fat pads from Sprague-Dawley rats (250-300 g) were removed and placed in Tyrode's basal salt solution (pH 7.2) at 00, thoroughly minced, and placed in siliconized vials (three fat pads per vial) each containing 40 mg of crude collagenase (Worthington Biochemicals), 6 mg of bovine-serum albumin, and 6 ml of Tyrode's balanced salt solution. The mixture was incubated at 370 for 45 min on a wrist action shaker and centrifuged in siliconized conical tubes (30 X g for 2 min). The resulting pellet contained larger blood vessels, stromal cells, and attached fat cells, and was discarded. The supernatant containing the fat cake was decanted and centrifuged (800 X g for 2 min). The vascular pellets from the second centrifugation were twice pooled and washed in Tyrode's solution, and collected by centrifugation at 800 X g for 2 min. T...

Glucose-6-phosphatase activity was demonstrated histochemically in rat liver using either the Wachstein-Meisel medium or a modified Chiquoine medium, and the characteristic properties of the enzyme activity were confirmed. The distribution of activity in both unfixed and hydroxyadipaldehyde-fixed material was demonstrated with the electron microscope. Activity was found in both smooth- and rough-surfaced elements of the endoplasmic reticulum of hepatic cells, including the nuclear envelope, but was absent from the plasma membrane. These findings further implicate the endoplasmic reticulum as an organelle of transport, and in addition suggest that the nuclear envelope has functional as well as morphological continuity with the endoplasmic reticulum.