Histochemical Study of a Fluoride Resistant Acid Phosphatase Reaction in the Mouse Duodenum

Burt, Robert C.; Meredith, Barbara R.; Grauer, Robert C.

doi:10.1177/5.2.135

Cited by 14 publications

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“…The dense areas do not represent a non-specific take-up of lead by protein, for the effect was not seen after floating the sections on lead nitrate or phosphate solutions. Nevertheless, the high concentration of sodium fluoride needed to inhibit the reaction suggests that the enzyme located may be alkaline rather than acid phosphatase, since the former is less sensitive to sodium fluoride (4,22). More work is required, possibly with other histochemical methods, before the true location of acid phosphatase can be established, but this investigation has shown the feasibility of using freeze-substituted sections for electron microscopic histochemistry.…”

Section: Discussionmentioning

confidence: 99%

The Staining of Thin Sections of Mouse Pancreas Prepared by the Fernández-Morán Helium Ii Freeze-Substitution Method

Bullivant

1960

The Journal of Cell Biology

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Small pieces of mouse pancreas were rapidly frozen in helium II, substituted in methanol at --75 ° C., and embedded in methacrylate by ultraviolet polymerization in the cold. The unstained cells show a structure similar to that after OsO4 fixation, except that the RNP particles have little or no contrast and the mitochondria and Golgi zones appear as grey areas without internal structure. After staining the sections by floating them on solutions of lead acetate or osmium tetroxide, there is an increase in contrast of RNP particles, ergastoplasmic membranes, and zymogen granules. Mitochondrial and Golgi membranes, zymogen granule membranes, and a membrane along the outside of the ergastoplasmic cisterna appear in negative contrast. The structure of the ergastoplasm, the existence of RNP particles, and the production of negative contrast are discussed. A modification of Gomori's method for acid phosphatase produces a lead deposit around the periphery of the zymogen granules. Possibly this deposit does not represent the true site of the enzyme, but the results show the feasibility of histochemistry at the level of resolution of the electron microscope.

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Section: Discussionmentioning

confidence: 99%

The Staining of Thin Sections of Mouse Pancreas Prepared by the Fernández-Morán Helium Ii Freeze-Substitution Method

Bullivant

1960

The Journal of Cell Biology

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Mucosal enzymes in the cecum of conventional and germfree mice

Jervis

Biggers

1964

Anat. Rec.

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The morphology of the cecal mucosa and the topographic distribution of certain mucosal enzymes were compared i n conventional and germfree mice to investigate the phenomenon of cecal enlargement observed in germfree animals. The cecal mucosa is predominantly non-villous in the conventional, but has a villous structure in the germfree mice. Alkaline phosphatase, in the conventional mice, is present in the striated border of a limited number of the epithelial cells lining the cecum; in the germfree mice almost all these epithelial cells show enzymatic activity. Acid phosphatase is more abundant in the epithelial cells and macrophages of the cecal mucosa of the conventional than of the germfree animals. Both groups show similar monoamine oxidase and reduced diphosphopyridine nucleotide diaphorase activity.The reduction of the villi, and the decrease in alkaline phosphatase activity in the conventional mice point to an involution of the mucosa as an absorptive organ. Barka ('63) has suggested that acid phosphatase may have a defensive function; its increase in the conventional animal would support this interpretation. The two oxidative enzymes studied apparently are not influenced by the intestinal flora.

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Localization of acid phosphatase in limb tissue of the adult newt, Diemictylus viridescens

Schmidt

1963

J. Exp. Zool.

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Acid phosphatase has been demonstrated histochemically in several mammalian tissues since Gomori introduced his method in 1941. Although surprisingly little information has been obtained about acid phosphatase in adult Urodele tissues, Kambara ('55) has demonstrated phosphatase reactivity in the epidermis, dermis, subcutaneous glands, and subcutaneous muscle, as well as in the urogenital system (Kambara, '56a, '56b) of adult Triturus pyrrhogaster.In our studies on the regenerating adult newt limb, we surveyed the normal tissues for acid phosphatase reactivity, applying both the lead nitrate method of Gomori ('50), and the azo dye technique devised by Burstone ('58). Observations were made on epidermis, connective tissues, subcutaneous glands, blood vessels, blood smears, nerves and striated muscle. The results of these observations are unique in several respects, and serve as a basis for further investigation. MATERIALS AND METHODSThe adult newt, Diemictylus viridescens, collected in the vicinity of Petersham, Massachusetts, served as experimental animals in this study.The humerus of the forelimb was surgically removed as described by Schmidt ('62a), and the limbs sampled post-operatively from day 1 through day 52.One series of 128 limbs were quickfrozen and substituted in acetone chilled to about -70°C with dry ice. After five days, the tissues were paraffin embedded at 54°C in vacuo for 30 minutes. Seven micron serial sections were mounted on coverslips with albumen-water, dried for four hours (45"C), and placed in a deep freeze at -20°C until staining the following day.A second series of 82 limbs were quickfrozen on a backing of beef muscle, either in liquid nitrogen, isopentane chilled to about -160"C, or in a cryostat at -20°C. Tissues were sectioned at 8 I-I in a cryostat, and mounted on coverslips without the use of adhesives. The mounted sections were placed in a dry-cabinet for one to 24 hours, and fixed in cold acetone (4°C) for 15 to 60 minutes prior to staining.Smears of adult newt blood were prepared and stained for acid phosphatase without prior fixation.Paraffin embedded sections were incubated for up to six hours at 37°C in a Gomori ('50) medium containing sodium B-glycerophosphate as substrate, buffered with .05 M sodium acetate to pH 4.9 (tests were conducted on liver sections at .5 pH unit intervals between pH 4.5 and pH 6.5). Sections which were deparaffinized following their incubation period (Goetsch and Reynolds, '51) showed sharper cytoplasmic localization and little nuclear activity.Two hours incubation at 37°C served to localize acid phosphatase reactivity in cryostat-cut sections.Naphthol AS-MX and naphthol AS-TR (Nutritional Biochemicals Corporation) served as substrates for the simultaneous coupling azo dye method of Burstone ('58). Fast blue salt BBN (General Dyestuff Co.) formed an insoluble blue precipitate at the site of enzyme activity. Incubation media buffered at pH 5.2 (tests at other pH levels were conducted: see above) with .2 M sodium acetate were freshly prepared as neede...

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Histochemical Study of a Fluoride Resistant Acid Phosphatase Reaction in the Mouse Duodenum

Cited by 14 publications

References 0 publications

The Staining of Thin Sections of Mouse Pancreas Prepared by the Fernández-Morán Helium Ii Freeze-Substitution Method

The Staining of Thin Sections of Mouse Pancreas Prepared by the Fernández-Morán Helium Ii Freeze-Substitution Method

Mucosal enzymes in the cecum of conventional and germfree mice

Localization of acid phosphatase in limb tissue of the adult newt, Diemictylus viridescens

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