A new method of urinary stone analysis by batik histochemical staining of thin sections.

Thin sections of urinary calculi are prepared by petrographic methods using Araldite as the mounting medium. By covering the remaining part of the section with wax, an exposed segment of the section is stained by a histochemical technique. By the process of dewaxing and rewaxing, successive adjacent segments are stained by GBHA, Von Kossa, Schultz, and titan yellow methods for calcium oxalate, apatite, uric acid and urates, and magnesium in magnesium ammonium phosphate, respectively. If desired, matrix in additional segments is stained with PAS and aqueous toluidine blue. Microscopic examination of each layer through all the stained segments of a stone section reveals its chemical nature. Thus the chemical composition, morphology, and spatial distribution of the crystalline and matrix constituents of thin sections of urinary calculi are simultaneously revealed in situ.     

Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postosmication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Physical, X-ray diffraction and scanning electron microscopic studies of uroliths

Identification of chemical constituents of calculus is important in the diagnosis and management of urolithiasis. The compositional variability of uroliths has different etiologies and requires various modes of treatment and prophylaxis. In the present study, we report the chemical compositional analyses of calculi recovered from buck and bullock by X-ray diffraction (XRD) and energy dispersive X-ray (EDX) techniques and ultra-structure examination by scanning electron microscopy (SEM). XRD and EDX investigations conclusively established the chemical compositions of urinary calculi under investigation. The calculus from buck (sample I) had calcium oxalate monohydrate, a dominant salt phase and magnesium compound in significant amount. The calculus from bullock (sample II) had magnesium ammonium phosphate phase, with significant amount of calcium in apatite form and K+ ions. SEM study at higher magnification (X 1000) showed bipyramidal crystals in external zones of urolith (sample I). The struvite apatite calculus showed that basic unit of structure was lamination and the laminitis appeared to be made up of fine granules and high porosity. The bio-mineralization process of calculus formation was also studied, with a view to take preventive and therapeutic measures for amelioration of urinary stone diseases in animals and humans.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Summary Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postomication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Demonstration of lipofuscin and Nissl bodies in crystal violet stained sections using a fluorescence technique or pyronin Y stain

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures--Nissl bodies and lipofuscin--can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.     

Immunocytochemical localization of cathepsin H in rat kidney

Summary Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For ligh microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Immunocytochemical localization of cathepsin H in rat kidney. Light and electron microscopic study

Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For light microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Use of the Four-and-a-Half Clearing Technique to Study Gymnosperm EmbryoIogy: Cunninghamia lanceolata

Since its introduction in 1971, the four-and-a-half clearing technique has been widely applied to the study of ovule and female gametophyte development in flowering plants as an alternative to the more arduous paraffin section methods. The technique has undergone several modifications that have broadened its application in studies of Angiosperm embryology. To date, however, the technique has not been successfully applied to embryological features of Gymnosperms. Dark coloration caused by naturally occurring substances and by-products of fixation render the clearing fluid ineffective, and special pretreatment methods used to remove dark substances in Angiosperm ovules have little or no effect on Gymnosperm material. In the technique reported here, paraffin sections of ovules and young seeds of Cunninghamia lanceolata 80-120 μm thick are cleared in benzyl benzoate-412 clearing fluid and examined with phase contrast optics. Observations of the mature female gametophyte in these cleared preparations are compared with those obtained from 10 μm sections, stained with safranin and fast green, and examined with bright-field optics. Although contrast and definition are more pronounced in stained sections than in cleared ones, the differences would not alter one's interpretation of characteristic structural features. The thick, cleared section offers an advantage over the thin, stained one in that many structural entities are contained within a single section rather than spread through several serial sections. The time required for clearing thick sections is much shorter than that required for making permanent stained preparations.     

Demonstration of Lipofuscin and Nissl Bodies in Crystal Violet Stained Sections Using a Fluorescence Technique or Pyronin Y Stain

This paper presents two simple, reliable methods for identification of lipofuscin and Nissl bodies in the same section. One method shows that lipofuscin stained with crystal violet retains its ability to fluoresce and can be observed under the fluorescence microscope after the stain has faded. Fading is accompanied by a gradual increase in the intensity of the fluorescence and is complete in about 5 min. Exciting illumination from this part of the spectrum also substantially fades staining of other autofluorescing tissue elements, such as lipids. Nonfluorescing structures, such as Nissl bodies, remain stained. By changing from transillumination with tungsten light to epifluorescent illumination and vice versa, both types of structures—Nissl bodies and lipofuscin—can be identified in the same section. The second technique uses pyronin Y for staining Nissl bodies in preparations previously stained with crystal violet. Nissl bodies are stained pink but lipofuscin remains violet. Lipofuscin in these sections also remains autofluorescent after the crystal violet stain has faded under violet or near-UV light.     

Quantitative microphotometric succinate dehydrogenase histochemistry in human nephron

A simple method for microphotometric evaluation of cryostat sections from human renal tissue routinely stained for succinate dehydrogenase activity by means of tetranitro-blue tetrazolium chloride is described and tested for validity. Manual absorbance measurement within single nephron segments from the same section allows to directly visualize the distribution pattern of this enzyme along the nephron. Photometric data can be expressed in relative enzyme activities by using the cortical collecting ducts within the same section as reference. This allows to compare measurements of different kidney sections stained by various incubation procedures. The agreement found between relative succinate dehydrogenase activities and recently published morphometric data on mitochondrial inner membranes along the rat nephron suggests that quantitative succinate dehydrogenase microphotometry is a useful histochemical tool for the assessment of renal mitochondrial cristae membranes.     

Renal Calculi

The pathogenesis of renal calculi is reviewed in general terms followed by the results of investigation of 439 patients with renal calculi studied by the author at Toronto General Hospital over a 13-year period. Abnormalities of probable pathogenetic significance were encountered in 76% of patients. Idiopathic hypercalciuria was encountered in 42% of patients, primary hyperparathyroidism in 11%, urinary infection in 8% and miscellaneous disorders in 8%. The incidence of uric acid stones and cystinuria was 5% and 2% respectively. In the remaining 24% of patients in whom no definite abnormalities were encountered the mean urinary magnesium excretion was less than normal. Of 180 patients with idiopathic hypercalciuria, only 24 were females. In the diagnosis of hyperparathyroidism, the importance of detecting minimal degrees of hypercalcemia is stressed; attention is also drawn to the new observation that the upper limit of normal for serum calcium is slightly lower in females than in males. The efficacy of various measures advocated for the prevention of renal calculi is also reviewed. In the author''s experience the administration of thiazides has been particularly effective in the prevention of calcium stones. Thiazides cause a sustained reduction in urinary calcium excretion and increase in urinary magnesium excretion. These agents also appear to affect the skeleton by diminishing bone resorption and slowing down bone turnover.     

A Rapid Marchi Technic

By varying the thickness of the nervous tissue immersed in chlorate-osmic-formalin staining fluid (Swank and Davenport, 1935) it was found that a section 1 mm. thick can be completely and adequately stained in 24 hours. Thicker sections require a proportionately longer time. The quality of the Marchi stain in the rapidly prepared section is as good as that in the material stained for 10 days although the background is slightly lighter in the latter preparations. This method can be used where time is an important element and is especially applicable to spinal cord, small animal brains, or portions of larger brains in which serial sections are not required.     

A Rapid Marchi Technic

By varying the thickness of the nervous tissue immersed in chlorate-osmic-formalin staining fluid (Swank and Davenport, 1935) it was found that a section 1 mm. thick can be completely and adequately stained in 24 hours. Thicker sections require a proportionately longer time. The quality of the Marchi stain in the rapidly prepared section is as good as that in the material stained for 10 days although the background is slightly lighter in the latter preparations. This method can be used where time is an important element and is especially applicable to spinal cord, small animal brains, or portions of larger brains in which serial sections are not required.     

A Method for Applying Different Stains to Alternate Serial Sections On a Single Microscope Slide

Every other section on the slide is coated with an inert grease (Dow Corning 7 Compound). The first staining technique is applied, thus staining the sections not covered with grease. The grease is dissolved in xylene, the slide rehydrated, and the sections already stained are coated with grease. The second staining technique is applied, and the grease is again dissolved in xylene, yielding finally a slide on which alternate sections have been stained by the two different staining techniques. Silicone greases, applied with a syringe and blunt hypodermic needle, are useful coating materials because of their insolubility in water and alcohol, chemical inertness, and heat stability. Silicone coatings are compatible with most staining techniques, including those which use strong oxidants, reductants, acids, bases, enzymes, and high temperatures. The method is particularly useful in examining serial sections of small blocks of tissue, or of tissues varying significantly in structure every few sections.     

Storage of glycoprotein in NCTR-Balb/C mouse. Lectin histochemistry, and biochemical studies.

A strain of Balb/C mice carrying a lysosomal storage disorder exhibits metabolic and phenotypic abnormalities similar to patients with sphingomyelin-cholesterol lipidoses type II (i.e., Niemann-Pick C and D). Their foamy cells, which belong to the reticuloendothelial system, stained intensely by periodate-Schiff (PAS) reagent and were resistant to predigestion with diastase. To identify the chemical nature of the PAS-positive storage material, we applied lectin histochemistry and biochemical methods. Paraffin embedded sections, and delipidated frozen tissue sections, were treated with biotinylated lectins and localized with avidin-biotin-peroxidase complex. Araldite-embedded semithin sections were incubated with biotinylated lectins followed by avidin-gold and were enhanced with silver. By both histochemical methods the affected foamy cells stained positively as follows: Concanavalia ensiformis agglutinin, Datura stramonium agglutinin, Griffonia simplicifolia-I, Lens culinaris agglutinin, peanut agglutinin, Ricinus communis agglutinin-I, wheat germ agglutinin (WGA), and succinylated-WGA. Biochemical analysis of liver extracts complemented the histochemical data and demonstrated accumulation of glycoproteins containing polylactosaminoglycans in affected mice. Our findings indicate that the storage material in NCTR-Balb/C mice is heterogeneous. The lipids that are extracted by organic solvents during the histologic preparations mask the occurrence of polylactosaminoglycan containing glycoproteins in native frozen sections.     

Urolithiasis in ferrets (Mustela putorius).

Urinary calculi was observed frequently in ferrets which were from a group used for influenza research. They were submitted for necropsy with various clinical signs. The calculi were composed of magnesium ammonium phosphate hexahydrate and were found in the pelvis of the kidney, urinary bladder and urethra. Crystals of undetermined nature occasionally were observed in the kidneys.     

Luxol Fast Blue as a Selective Stain for Alpha Cells in the Human Pituitary

Human pituitaries fixed in Bouin's fluid or 10% formalin were stained by the PAS, Masson trichrome and luxol fast blue methods. By comparing adjacent sections stained by these 3 methods it was found that the alpha cells which are PAS negative, but stained red by the Masson trichrome method, were intensely stained by luxol fast blue. The beta cells which are stained blue by the PAS and Masson methods were not stained by luxol fast blue. Similar observations were made on a series of pituitaries from 8 other mammalian species. It is concluded that luxol fast blue is a selective stain for alpha cells in the mammalian pituitary.     

Reliable diagnosis of Trichosomoides crassicauda in the urinary bladder of the rat

Two reliable methods are described for identifying infection of laboratory rats with the nematode Trichosomoides crassicauda. The first is a rapid method where cryostat sections of the rat urinary bladder are stained with acridine orange and viewed under a fluorescence microscope. The second involves the stabilization of the bladder surface prior to examination using scanning electron microscopy (SEM).     

Drug-induced urinary calculi

Urinary calculi may be induced by a number of medications used to treat a variety of conditions. These medications may lead to metabolic abnormalities that facilitate the formation of stones. Drugs that induce metabolic calculi include loop diuretics; carbonic anhydrase inhibitors; and laxatives, when abused. Correcting the metabolic abnormality may eliminate or dramatically attenuate stone activity. Urinary calculi can also be induced by medications when the drugs crystallize and become the primary component of the stones. In this case, urinary supersaturation of the agent may promote formation of the calculi. Drugs that induce calculi via this process include magnesium trisilicate; ciprofloxacin; sulfa medications; triamterene; indinavir; and ephedrine, alone or in combination with guaifenesin. When this situation occurs, discontinuation of the medication is usually necessary.