Histochemical demonstration of norepinephrine in postmortem tissues

Summary A modified fluorescence method for the demonstration of norepinephrine in cryostat sections may be used with postmortem tissues and surgical specimens stored for as long as 24 hours at 4° C., and 6 hours at room temperature. Quantitative assay for norepinephrine in these tissues show no significant loss of catecholamine during such storage.Supported in part by Grant HE 10465 from the National Institute of Health.     

Eosinophilic leucocytes and phospholipase B of rat tissues

The correlation between the eosinophilic leucocyte population and the phospholipase B activity of rat tissues has been tested with isolated cell preparations from intestine, lung, blood, bone marrow and spleen containing eosinophils in varying proportions and with pure eosinophil fractions separated by centrifugation on discontinuous metrizoate and metrizamide gradients. A uniform value of activity per cell was found in all these tissues extending previous histochemical and biochemical evidence that the eosinophil is the carrier cell for the phospholipase B to all major sites of distribution. The enzyme marker approach has been used for estimating the normal eosinophil population of rat organs and show the distribution pattern of the eosinophils in peripheral organs in the wake of increased production and release from the marrow.     

Histochemical demonstration of rat liver glycogen phosphorylase activity with iron (Fe++)

Summary A new histochemical method for light microscopic demonstration of liver glycogen phosphorylase activity has been introduced in this study.The method demonstrates phosphorylase activity by precipitating phosphate ions, liberated in the reaction catalyzed by the enzyme, with Fe⁺⁺ present in the incubating medium. The precipitate is visualized as ferrous sulphide.The new glycogen, formed in the same reaction, can also be demonstrated in this method after staining with iodine.The lobular localization of the reaction products obtained according to this method in the liver, corresponds well to that obtained according to earlier methods for the demonstration of only new-formed glycogen.     

The demonstration of a specific 5'-nucleotidase activity in rat tissues.

5′-Nucleotidase has been partially purified from rat liver, spleen, kidney, heart, lung, brain and skeletal muscle. The majority of the enzyme activity in each of these tissues was insoluble in 1% of Triton X-100, solubilized in 2% Triton X-100,1% sodium deoxycholate, and stable to incubation at 50 °C for 5 min. The partially purified enzyme from each tissue exhibited the same pH optimum, was inhibited by concanavalin A, and was inhibited in an identical manner by antibody to highly purified 5′-nucleotidase from liver. Since the enzyme is usually concentrated in the plasma membrane (De Pierre, J. W. and Karnovsky, M. L. (1973) J. Cell Biol., 56, 275–303), the results indicate that the enzyme may represent a convenient and general marker for this organelle in rat tissues.     

Histochemical demonstration of mercury induced changes in rat neurons.

A histochemical method modified for ultrastructural studies of mercury induced changes is described. Rat neurons from areas known to be influenced by mercury are used as examples. The histochemical reaction, suggested to be caused by polymercury sulphide complexes, is localized to "dense bodies" where it is visible 14 days after initiation of peroral mercury treatment (20 mg HgCl2/l drinking water).     

Histochemical demonstration of carbonic anhydrase activity

Summary Freeze-dried frozen sections are floated on the surface of the freshly prepared incubation mixture (CoSO₄ 1.75 × 10^–3 M, H₂SO₄ 5.3 × 10^–2 M, NaHCO₃ 1.57 × 10^–2 M and KH₂PO₄ 1.17 to 11.7 × 10^–3 M; demonstration of weak activity requires high phosphate). A compound containing cobalt and phosphorous precipitates at carbonic anhydrase sites and is converted to CoS. Adequate staining requires only 2–10 minutes of incubation. Actazolamide inhibits the staining reaction in specific concentrations. Actazolamidein vivo, 20 mg/kgi.v. to mice 30 minutes before sacrifice also inhibited the staining. The proportion phosphorous in the specific precipitate increases with KH₂PO₄ of the medium (shown by the addition of⁶⁰Co and³²P). An explanation of the reaction mechanism is given, based on the catalyzed loss of CO₂ in the surface layer. The inclusion of phosphate in the medium makes this modification ofHäusler's method so sensitive that it shows carbonic anhydrase activity in for instance stratum spinosum of the skin.This investigation was supported by grants from the Medical Faculty, University of Uppsala and from the U.S. National Institutes of Health (Grant NB 3060 to E.Bárány).     

Histochemical demonstration of copper in LEC rat liver

Livers of LEC rats were histochemically stained for copper according to the modified Timm's method, which includes trichloroacetic acid (TCA) treatment. TCA pretreatment was effective in removing zinc and iron, leaving copper as the major metal in the liver. Hepatocytes in 3-month-old rats were stained intensely by the modified Timm's method, both in frozen sections and in paraffin-embedded specimens. The centrilobular hepatocytes were usually stained, but positive cells were also randomly distributed in the hepatic lobes, showing a mosaic pattern. The staining was intensified in 8- compared to 3-month-old LEC rats. In contrast hepatocytes from LEA rats, the normal counterpart of LEC rats, were faintly stained for copper. Proliferating cholangioles found in older LEC rats were shown to lack copper deposition, and hepatocellular carcinoma showed less copper deposits than the hepatocytes surrounding the tumor. The copper staining was augmented in livers of LEC rats subjected to copper-loading, but was less intense in the livers treated with d-penicillamine. The staining intensity under the various experimental conditions showed good correlation with the copper concentration. Lysosomal deposition of copper in hepatocytes was demonstrated by electron microscopic analysis for copper. Thus the modified Timm's method was shown to produce valuable results in demonstrating copper in LEC rat livers, providing important information for an understanding of the mechanism of copper deposition and hepatic disease of the animal.     

Light and electron microscopic demonstration of phospholipase B activity in the mouse eosinophil

Phospholipase B (EC 3.1.1.5) which hydrolyzes phospholipids in the alpha and beta positions was demonstrated in murine leukocytes using light and electron microscopic histochemical techniques. Leukocytes (neutrophils, lymphocytes, macrophages, eosinophils) were harvested from peritoneal exudates of mice. Cells were fixed in 4% calcium-formol fixative for 10 min at 4 degrees C for light microscopy and 30 min at room temperature for electron microscopy, after which they were incubated at 37 degrees C in medium at pH 6.6 containing 2 microM lysolecithin and CaCl2. The fatty acids released during the hydrolytic reaction were trapped as a calcium precipitate and were converted to a cobalt precipitate for light microscopy by treatment with cobalt acetate or to a lead precipitate for electron microscopy by treatment with lead nitrate. The reaction products were observed to be present in eosinophils and absent in neutrophils, lymphocytes and macrophages. It is concluded that the eosinophilic leukocyte is the carrier cell for phospholipase B in inflammatory reactions.