Journal of Histochemistry & Cytochemistry

The preparation and characterization of a horseradish peroxidase-rabbit antiperoxidase Fab immunocomplex (HRP-Fab2) useful for immunocytochemical localization of primary tissue-bound rabbit antibody are described. Antisera with titer to horseradish peroxidase (HRP) were raised in rabbits. Anti-HRP-Fab fragments were prepared by controlled mercuripapain digestion of the purified rabbit IgG. The complex was formed during incubation of Fab fragments with HRP, and fractions containing HRP activity that were precipitable by goat anti-rabbit IgG serum were isolated by gel filtration. The major isolated complex had a molecular weight of approximately 150,000 daltons and migrated as a single band on cellulose acetate electrophoresis. Polyacrylamide gel electrophoresis in SDS indicated the major polypeptide components of the complex were HRP and Fab. RZ (absorbance at 403 nm/275 nm) determination indicated a molar ratio of 2 Fab:1 HRP. The complex was stable for at least 1 year at -20 degrees C and was used successfully in a number of immunocytochemical procedures.

Cytochemical evidence for the existence of a Golgi-associated phosphatase activity that hydrolyzes nicotinamide adenine dinucleotide phosphate (NADP) at acid pH in rat incisor ameloblasts was obtained by incubating sections from glutaraldehyde-fixed teeth in a medium containing NADP as substrate and lead ions as capture agent. Following incubation for 1 hr at 37 degrees C and pH 5.0, the Golgi saccules situated between those at the cis (immature) and trans (mature) faces of the ameloblast Golgi apparatus were marked by reaction product with the heaviest deposit in the middle saccule. Reaction product was otherwise seen in trace amounts only over some elements of the GERL system as well as a few lysosomal dense bodies and immature secretory granules. Control experiments established that the selective staining of intermediate Golgi saccules at pH 5.0 could only be duplicated by using substrates that resembled the complete NADP molecule, and not just the portion containing the adenosine 2'-monophosphate group. As well, no deposits of reaction product were seen within the Golgi saccules of ameloblasts incubated at pH 5.0 with nictoinamide adenine dinucleotide (NAD) as the substrate or that were incubated at pH 7.2 or pH 9.0 with NADP as the substrate. It was concluded that a specific, acid-NADPase activity is present in the intermediate Golgi saccules of secretory ameloblasts. Preliminary observations on other cells suggest that the localization of NADPase activity to Golgi saccules may constitute a general phenomenon.

Cytochemical tests for nucleosidetriphosphatase (NTPase) and Bernhard's preferential staining for ribonucleoproteins (RNP) were applied to isolated rat liver nuclei. The strongest and most easily reproducible positive reaction for NTPase was detected at pH 7.7 with ATP and GTP. This reaction was activated by Mg2+ and Ca2+ and inhibited by Be2+, Zn2+, quercetin, and ribonuclease. The major sites of enzyme reaction were intranuclear RNA-containing structures. Incubation of nuclei in ATP-stimulated RNA-release medium eliminated a considerable part of the material showing both NTPase reaction and staining for RNP; the perichromatin granules disappeared, while interchromatin granules remained. NTPase activity in the nuclear envelope seems to be associated with the annular part of nuclear pore complexes (permanent component) and with RNP particles translocated through nuclear pores or attached to the surface of nuclei (transitional component). From a morphological point of view, these observations support previous biochemical data suggesting the existence of a connection between NTPase activity and the translocation of RNP particles through the nuclear envelope.

Data are presented that show that a histochemical stain, Hoeschst 33342, can be used to discriminate between viable B and T lymphocytes in the mouse. Quantitative analysis of the staining of cells from various lymphoid tissues with Hoechst 33342 using a Fluorescence-Activated Cell Sorter (FACS) indicates that two populations of cells can be identified. In the spleen approximately 60% of the lymphocytes can be classified as brightly stained with 1 microgram/ml of Hoechst 33342, while in the lymph node only 40% of the cells stain brightly. Thymocytes exhibit only the dull staining profile. Separation of these two populations from the spleen using the FACS and reanalyzing them for cell surface antigenic markers shows that the lymphocytes stained brightly with Hoechst 33342 are predominantly immunoglobulin positive, while the cells that stain less brightly express Thy 1.2. This indicates that a histochemical stain correlates directly with classical immunological markers on cell surfaces.

The sensitivity of Ilford L4 emulsion to 45Ca was determined for electron microscope autoradiographic conditions. Sensitivity values were obtained for monolayers and double layers of emulsion in combination with various developing procedures. The dependence of sensitivity upon radiation dose was determined. All results are compared to previously calibrated isotopes.

The cell membrane and X bodies of histiocytes X from two cases of eosinophilic granuloma were stained by nonspecific esterase. The results show that histiocytes X possess a cell membrane exoenzyme similar to that of other histiocytes, such as alveolar macrophages, but the role of the X body as related to enzyme activity remains an unknown.

A device is described for the rapid freezing of tissue in situ by a punch biopsy approach using a specially designed cryogun with a highly thermal conductive specimen holder. The cryogun consists of a sampling device using a double, spring-loaded gun mechanism and a system of cryochambers. Ultrathin freeze-dried sections cut from samples obtained with this cryogun are relatively free of artifacts and have few ice crystals. Organelles are seen by natural contrast when cryosections of approximately 1000 A are observed with a transmission electron microscope or in the transmission mode of a scanning electron microscope. The construction of the cryogun is described along with a method of obtaining improved, ultrafast cryofixation of tissue specimens. The reliability of obtaining x-ray microanalysis measurements of diffusible ions where movement within cell compartments has been retained is discussed.

The effect of trypsin digestion on the immunoperoxidase-PAP staining of paraffin sections from normal and abnormal tissue was studied. Trypsin treatment reduced background staining considerably, but enhanced specific staining. Results were, therefore, much easier to read. Possible reasons for this result are discussed.

This study examined 1) the effect of lead and fixation on adenylate cyclase activity, 2) the effect of lead on App(NH)p, and 3) the specificity of App(NH)p as a substrate for adenylate cyclase under the conditions of the cytochemical assay. The results indicated that: 1) fixation that provides adequate structural preservation inhibits enzyme activity to varying degrees depending on the tissue, fixative, length and temperature of fixation; 2) millimolar concentrations of lead do not negatively affect the adenylate cyclase activity of several different tissues (especially if 10 mM NaF is present); 3) lead does not cause the nonenzymatic hydrolysis of App(NH)p; 4) the App(NH)p obtained from the supplier is contaminated and should be purified before use, since lead can interact with the contaminants and this may be a source of error in the assay; and 5) adenylate cyclase appears to be the major enzyme that cleaves App(NH)p under cytochemical conditions.

Problems encountered in the histochemical localization of alkaline phosphatase activity are discussed and solutions presented. The purpose is to achieve a reaction that can be studied by light and electron microscopy and to distinguish alkaline glycerophosphatase from inorganic pyrophosphatase activity. Details are presented concerning fixatives, fixation times, incubation media, enzyme inhibitors, activators, and associated techniques that can be used to obtain optimal histochemical results.

The main development in ATPase cytochemistry in recent years is the development of three methodologically independent approaches to the localization of Na+, K+-ATPase. These methods in general support one another well and have led to significant insights into the mechanisms of bulk cation transport at several important sites. The area most in need of development is now the problem of specific localization of divalent ion-dependent ATPases at the plasma membrane. The value of the traditional Wachstein-Meisel methods appears to be very limited, and progress will depend on the introduction of more specific techniques.

The freeze-dry autoradiographic method devised originally by Stirling (J Cell Biol 53:704, 1972) to localize Na+ pump sites with (3H)ouabain is reviewed. Biochemical, physiological, and autoradiographic data are discussed which establish that ouabain binding to intact tissue conforms to rigid criteria for high Na+ pump specificity. Among these are that glycoside binding exhibits saturation kinetics, ligand dependence, and close correlation with degrees of inhibition of Na+-K+-ATPase and Na+ transport. Moreover, localization of Na+ pump sites by this technique shows a cell and membrane specificity which mirrors that obtained by cytochemical and immunocytochemical methods. In addition to resolving cell-specific patterns of localization in heterogeneous tissues, the demonstration of Na+-K+-ATPase by these techniques indicates that Na+ pumps are distributed uniformly along plasmalemmal surfaces and are restricted to the basolateral interface in reabsorptive and secretory epithelia despite the opposing polarity of net transepithelial electrolyte transport.

Acid phosphatase activity, a lysosomal marker, is commonly demonstrated using the Gomori technique with cytidine 5'-monophosphate or beta-glycerophosphate as substrate. Using this lead capture method on mouse and rat exorbital lacrimal, parotid, and pancreatic acinar cells, reaction product was localized in GERL, forming secretory granules, and secondary lysosomes. However, a different cytochemical localization was observed for inorganic trimetaphosphatase, another lysosomal enzyme. When the technique for trimetaphosphatase activity, a metal chelation method, was applied to exocrine acinar cells, reaction produce was conspicuously absent from GERL and forming secretory granules, but was present in secondary lysosomes, occasionally in Golgi saccules, and in previously unreported basal elongated lysosomes. The differences in the localization of the two enzymatic activities emphasizes the importance of employing more than one substrate where possible, and raises questions concerning the mechanism of delivery of acid hydrolases to secondary lysosomes.

GERL exhibits a number of structural properties, such as its location at the trans face of the Golgi apparatus, thick limiting membranes with occasional coated regions, and close relationship to the ER, which are similar in all cells wehre it has been identified. Acid hydrolase activity and the formation of lysosomes, also considered characteristic of GERL, have been shown to be less consistent properties of GERL in some cells. Finally, GERL and the Golgi saccules of certain cells undergo significant changes in their cytochemical and structural properties in response to specific alterations in cellular activity. These variations are important for at least two reasons: first, they indicate the care required in differentiating GERL from the Golgi saccules based on limited cytochemical observations; second, and most important, they may yield information regarding the biogenetic and functional relationships between the ER, Golgi saccules, and GERL, such as the origin of GERL in various cells and the role of each organelle in the processing of lysosomal and secretory proteins.

The thiol ester, N-t-butyloxycarbonyl-L-alanine-p-nitrothiophenyl ester (Boc-Ala-SNp) has been synthesized and applied as an ultrastructural cytochemical substrate for leukocytic elastase-like enzymes. Incubation of fixed human neutrophils with Boc-Ala-SNp in the presence of gold ions generates electron-dense deposits of gold p-nitrothiophenolate in the nuclear membrane, endoplasmic reticulum, Golgi complex, mitochondria, and granules. Deposition of product is inhibited by pretreatment of cells with general and specific chemical inactivators of neutrophil elastase. This substrate appears to have significant potential as a probe for the ultrastructural localization of elastase-like activity.