Elastoid Changes in the Gingiva of Aged Humans

Elastotic changes were demonstrable in the gingivae of both dentulous and edentulous jaws obtained from both male and female humans varying in age from 62–92 years. Sections of gingivae from all the aged individuals exhibited numerable thick fibers that stained positive with iron hematoxylin or orcein. These positive staining fibers were found in the lamina propria radiating into the connective tissue papillae, coursing throughout the zona reticularis, as well as appearing as black amorphous masses. Pretreatment of sections with acid hydrolysis before staining by the two elastic tissue stains led to a loss of stainable fibers for elastin. In contrast the gingivae of a young adult did not contain elastic positive fibers as seen in the aged gingivae. The thick elastotic fibers found in the aged gingivae were argyrophilic in nature when the sections were impregnated with silver nitrate indicating that they were collagenous in nature. It is felt that the elastoid-like fibers in aging gingiva are another phase in the altering of collagen during the aging process.     

Alcoholic Bouin fixation of insect nervous systems for bodian silver staining. III. A shortened, single impregnation method

Satisfactory Bodian silver staining of paraffin wax sections of both locust (Schistocerca gregaria) and cockroach (Periplaneta americana) central nerve tissue can be obtained with only one impregnation, instead of the usual two, by the following modified procedure. Freshly dissected ganglia are fixed in an improved synthetic alcoholic Bouin (40% formaldehyde 0-15: ethanol 25: acetic acid 5: picric acid 0.5: either ethyl acetate 5 and diethoxymethane 15, or ethyl acetate 25: distilled water to 100). Formaldehyde content governs intensity of glial staining (little or none without formaldehyde) and the mixture with more ethyl acetate substituted for diethoxymethane gives more intense staining overall. Sections are impregnated once only, overnight, in 2% Protargol solution brought to about pH 8.4 with ammonium hydroxide and containing 1.3 g of copper per 65 ml. Depending on fixative composition, species, section thickness and contrast desired between nerve fibers and background, the subsequent distilled water rinse is shortened or omitted and sections are developed in 1% hydroquinone with sodium sulfite content reduced (to 2.5-4% Na2SO3.7H2O) for thinner (10 micrometer) sections but normal (10%) for thicker (20 micrometer) ones. Sections are finally washed, gold intensified, treated with sodium thiosulfate and dehydrated, cleared and mounted as usual. Results are slightly lighter than with normal double impregnation but entirely suitable for studies of neuroanatomy.     

Microwaves Applied to Silver Impregnations with Ammoniacal Silver Carbonate

Techniques for impregnation with ammoniacal silver carbonate provide valuable information on all types of tissue; however, the time investment required to impregnate a few sections has limited their application. We have shortened the impregnation times by using microwaves in techniques for reticular fibers, astrocytes, nerve fibers and chromaffin cells. The results were satisfactory with markedly reduced impregnation time and elimination of nonspecific silver deposits.     

大鼠肠系膜淋巴结内高内皮微静脉周围网状支架的研究

目的：研究高内皮微静脉周围网状细胞和网状纤维网的形态、密度、排列方式及其空间联系,探讨高内皮微静脉周围网状的组织构筑与功能。方法：用镀银染色光镜观察法和冻裂割断扫描电镜观察法观察健康、成熟Wistar大鼠肠系膜淋巴结内高内皮微静脉的基质网状结构。结果：高内皮微静脉周围基质结构主要由密集、交织的网状细胞和网状纤维组成,并在高内皮微静脉周围形成轮样结构。结论：高内皮微静脉周围网状纤维形成网状纤维网,并与淋巴迷路周围的淋巴迷路周围的网状纤维网相连,可能是淋巴细胞归巢的重要通路。     

Alcoholic Bouin Fixation of Insect Nervous Systems for Bodian Silver Staining. III. A Shortened, Single Impregnation Method

Satisfactory Bodian silver staining of paraffin wax sections of both locust (Schistocerca gregaria) and cockroach (Periplaneta americana) central nerve tissue can be obtained with only one impregnation, instead of the usual two, by the following modified procedure. Freshly dissected ganglia are fixed in an improved synthetic alcoholic Bouin (40% formaldehyde 0-15:ethanol 25:acetic acid 5: picric acid 0.5:either ethyl acetate 5 and diethoxymethane 15, or ethyl acetate 25:distilled water to 100). Formaldehyde content governs intensity of glial staining (little or none without formaldehyde) and the mixture with more ethyl acetate substituted for diethoxymethane gives more intense staining overall. Sections are impregnated once only, overnight, in 2% Protargol solution brought to about pH 8.4 with ammonium hydroxide and containing 1.3 g of copper per 65 ml. Depending on fixative composition, species, section thickness and contrast desired between nerve fibers and background, the subsequent distilled water rinse is shortened or omitted and sections are developed in 1% hydroquinone with sodium sulfite content reduced (to 2.5-4% Na₂SO₃·7H₂O) for thinner (10 μm) sections but normal (10%) for thicker (20 μm) ones. Sections are finally washed, gold intensified, treated with sodium thiosulfate and dehydrated, cleared and mounted as usual. Results are slightly lighter than with normal double impregnation but entirely suitable for studies of neuroanatomy.     

Effect of Temperature on Argyrophil Impregnation: Development of A High Temperature Rapid Argyrophil Procedure

Out studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

Effect of temperature on argyrophil impregnation: development of a high temperature rapid argyrophil procedure

Our studies on the effects of temperature on the demonstration of neurosecretory granules using argyrophil stains indicate an inverse relationship between the time needed for staining and temperature of the silver and reducing solutions. Careful monitoring of the temperature of silver solutions during the Grimelius procedure and its modifications show long incubation times serve in large part only to bring the solutions to reaction temperature. Tissue sections added when this temperature has been reached will stain with the same intensity as sections impregnated for the entire incubation period. We have modified the argyrophil procedure so that double-impregnation with solutions preheated to 60-70 C and development in Bodian's reducer prepared with preheated water rapidly demonstrates secretory granules. Our method does not require a microwave oven and much shorter incubation periods are required than with usual procedures. It is not necessary to incubate sections in hot solutions for extended periods of time, which can lead to detachment of sections, nonspecific staining and decomposition of the silver solution. Rinsing after impregnation and before development greatly increases contrast of argyrophil cells by reducing background staining. Our procedure results in more reliable staining of argyrophil and argentaffin cells and takes only ten minutes.     

A Method for Silver Impregnation of Mammary Myoepithelia

Histochemical methods for microscopic visualization of nummary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

A method for silver impregnation of mammary myoepithelia

Histochemical methods for microscopic visualization of mammary myoepithelial cells all yielded considerable variation in completeness of myoepithelial cell staining. Although extremely variable, silver impregnation occasionally gave tissue sections containing myoepithelia having excellent microanatomical detail and contrast with other tissue elements. Consequently, sources of variation in the silver technique were considered. Composition of the tissue fixative and pH of the silver impregnating solution were most critical. A final method is presented which gives consistent, complete silver impregnation of myoepithelia, where both the cell body and cell processes are clearly evident. The staining procedure is not light sensitive, nor is acid cleaning of glassware necessary. Tissue sections from lactating mouse, rat, hamster and goat are presented; tissue from other species should stain as well. The procedure should greatly facilitate the study of the function of myoepithelial cells and the visualization of these cells in mammary pathology.     

Production and characterization of a monoclonal antibody to human type III collagen

Human type III collagen from placenta was isolated and purified for use as an immunogen. A monoclonal antibody was produced which specifically recognizes epitopes unique to type III collagen. The specificity of the antibody was determined by inhibition ELISA, an immunoblot assay, and by immunoprecipitation. Results indicated that the monoclonal antibody recognized only the alpha 1(III) polypeptide chains and did not crossreact with type I, IV, or V collagen. The monoclonal antibody was also used for immunohistochemical localization of type III collagen in tissue sections of human placenta, bovine spleen, and lymph node. In placenta, both large and small blood vessels showed pronounced staining of the tunica media, which contains largely smooth muscle cells, known to synthesize type III collagen. In contrast, the intimal areas and endothelial cells showed no staining with the antibody. In the placental villi, staining was limited to the villous core, where fine fibrillar structures showed strong staining. In lymph nodes, the capsule and pericapsular adipose cells were surrounded by a covering of type III collagen. Within the parenchyma of the node, staining was localized to a branching, reticular array of fine fibers. In the spleen, staining was pronounced in the capsule, splenic trabeculae, and white pulp, where blood vessel staining was especially prominent. The red pulp and splenic sinuses contain little or no type III collagen. The fine network-like or reticular staining pattern found in the lymph node parenchyma is consistent with the staining pattern of the protein reticulin, and suggests that type III collagen may be closely associated with reticulin in certain tissues. Since the role of type III in tissues is unclear, this reagent will be useful in providing new information in this regard.     

Effects of Oxidation on Neurofibrillar Argyrophilia

The effect of oxidation on neurofibrillar argyrophilia was studied by subjecting nervous tissues containing both normal and degenerating fibers to the action of potassium permanganate, periodic acid, chromic acid, lead tetraacetate, and sodium bismuthate prior to silver impregnation. The argyrophilic response of normal fibers to such treatment was studied with the Nonidez silver nitrate block technic, the double impregnation method of Bielschowsky on both blocks and sections, and a silver proteinate procedure. The response of degenerating fibers was studied by the Cajal formula 6 block technic and the modified Bielschowsky procedure of Nauta and Ryan for sections. The experimental data indicated that such oxidation did not produce any differential staining effects between normal or degenerating fibers.     

A New Method for Easy Demonstration of Argyrophil Cells

A new method for silver impregnation of endocrine cells of the gastrointestinal mucosa is described. It offers great reliability, eveness of impregnation, and, since it can be used on batches of slides, is also suitable for histology class and investigation material. The procedure for paraffin sections of formalin-fixed material is as follows: dewax and transfer to distilled water, leave in 0.5% silver nitrate solution for 2 hours at 60 C. Rinse in distilled water, then treat in Bodian developer (hydroquinone, 1 g; sodium sulphite, 5 g; distilled water, 100 ml) previously heated to 60 C. Rinse in running tap water, distilled water, and then re-impregnate for 10 minutes at 60 C in the same silver solution and reduoc in Bodian's solution. Sma the background is not impregnated by this method, sections may be counterstained by any basic anilin dye to bring out nuclei. A 0.1% kernechtrot solution was found very satisfactory in this respect. The granulations of argyrophil cells stand out sharply black against a red background.     

A new method for easy demonstration of argyrophil cells.

A new method for silver impregnation of endocrine cells of the gastrointestinal mucosa is described. It offers great reliability, eveness of impregnation, and, since it can be used on batches of slides, is also suitable for histology class and investigation material. The procedure for paraffin sections of formalin-fixed material is as follows: dewax and transfer to distilled water, leave in 0.5% silver nitrate solution for 2 hours at 60 C. Rinse in distilled water, then treat in Bodian developer (hydroquinone, 1 g; sodium sulphite, 5 g; distilled water, 100 ml) previously heated to 60 C. Rinse in running tap water, distilled water, and then re-impregnate for 10 minutes at 60 C in the same silver solution and reduce in Bodian's solution. Since the background is not impregnated by this method, sections may be counterstained by any basic anilin dye to bring out nuclei. A 0.1% kernechtrot solution was found very satisfactory in this respect. The granulations of argyrophil cells stand out sharply black against a red background.     

Differentiation of Myofibrillae, Reticular and Collagenous Fibrils in Vertebrates

A procedure for the differentiation of the mesenchymal derivatives, myofibrillae, reticular and collagenous fibers is presented. Formol-Zenker fixation (5-12 hours) is followed by the washing, iodinization, dehydration and paraffin embedding steps routine for that fixative with the following modifications. Zirkle's butyl alcohol series is used for dehydration and infiltration with paraffin as well as in the alcohol slide series. Embedding paraffin used is Parawax plus 8-10% bayberry wax. Tissue-exposed surface of paraffin block is soaked in water overnight before cutting serial sections at 3-5μ. Sections are mounted using the dilute albumen method, and the slides, thoroughly dried at 37^oC. overnight, are left at 60^o for 10 minutes to melt the paraffin of the sections. Before staining, the sections are given a preliminary treatment with potassium permanganate and oxalic acid. For reticular staining a 10% silver nitrate bath is succeeded by an ammoniacal silver carbonate solution followed by reduction in 1% neutral formalin, toning in gold chloride and fixing in sodium thiosulphate. Myofibrillae, the sacroplasmic limiting membrane and other sarcous elements are stained by Heidenhain's azocarmine solution, adult tissues at room temperature and fetal tissues at 50 ^oC. Differentiation in phosphotungstic acid is followed by the staining of collagenous fibers. For adult tissue, light green SF (C.C.) is used and for fetal tissue, fast green FCF (C.C). A discussion of the preparation of ammoniacal silver solutions is included. Both stock and used solutions of ammoniacal silver have been in use by the author for over a period of two years.     

Histological Technic for Cerebellar Climbing and Mossy Fibers

In order to, avoid disadvantages attendant upon the use of fresh frozen sections, or of block impregnation with silver, in staining climbing or mossy fibers of the cerebellum, Rio Hortega's double impregnation method for nerve fibers is useful. This consists of prolonged formalin fixation prior to cutting frozen sections (which thereafter are easier to cut) and preliminary treatment with ammoniacal aqueous and alcoholic washes, mordanting in pyridine silver, and treatment with pyridine-silver-carbonate. Following this, sections are handled individually through one of several reduction methods after which they may be directly mounted or gold toned.     

Type V collagen in splenic reticular fibers of the macaque monkey

The distribution of type I, III and V collagens in the monkey spleen was examined by indirect immunofluorescent microscopy and immunoelectron microscopy, and compared with that of reticular fibers revealed by a silver impregnation method. Type I collagen was localized on reticular fibers in the white pulps and on coarse reticular fibers in the splenic cords. Type III collagen was localized on the reticular fibers in the white pulps, and on the coarse reticular fibers and a limited number of fine reticular fibers, in the splenic cords. The anti-type V collagen antibody reacted with annular reticular fibers around the splenic sinuses, as well as with the reticular fibers in the white pulps and with the coarse and fine reticular fibers in the splenic cords. Thus, the distribution pattern of fibers that reacted with the anti-type V collagen antibody was very similar to that of the reticular fibers revealed by the silver impregnation method. Electron-microscopically, the fine reticular fibers in the splenic cords were composed of collagen fibrils, 30-50 nm in diameter, and amorphous substances. They were covered by reticular cell processes. By immunoperoxidase labeling with the anti-type V collagen antibody, electron-dense reaction products were found over the collagen fibrils with a banding pattern. These results indicate that type V collagen is an indispensable component of the reticular fibers.     

Silver Impregnation of Nerve Fibers in Teeth After Decalcification With Ethylenediaminetetraacetic ACID

The difficulties in impregnating bony tissues, which occur after decalcification with acids or electrolysis are avoided by decalcification with ethylenediaminetetraacetic acid at pH 8.2-8.5. The decalcification of adult human teeth which have been cut to a thickness of 2-5 mm takes 1-2 mo. If frozen sections of the decalcified teeth are impregnated 24 hr in 20% AgNo₃, rinsed through 6 changes of 20% neutralized (CaCO₃) formalin, blotted thoroughly with a cloth and placed in an ammoniated silver solution for 15-20 min, reliable impregnation of nerve fibers is obtained. The stock ammoniated silver solution is prepared by adding concentrated NH₄OH to 10-20 ml of 20% AgNO₃ until the precipitate formed by it is dissolved and then adding a few drops of the silver solution until the first permanent opalescence of the mixture is obtained. From this 2 ml are diluted directly before use with 6 ml of distilled water and 4 drops of concentrated NH₄OH added. The diluted stock solution should be used for few (5-10) sections only. The rest of the technic is done in the routine manner.     

The nucleolonema changes its three-dimensional conformation during interphase in root-tip meristems of onion

Summary The three-dimensional architecture of a filamentous nucleolar structure, called the “nucleolonema”, was investigated in onion root-tip cells by applying a silver impregnation technique to air-dried cells and serial ultrathin sections. The entire configuration of the nucleolonema was revealed when silver staining was applied to air-dried cells. The nucleolonema was knobbly or segmented along its entire length and showed great variation in thickness. Three categories of nucleolonema were discriminated depending on thickness; each had an average value of 0.5, 1.0, and 1.3 μm, respectively. Some root tips were embedded in Lowicryl K4M resin and cut into serial ultrathin sections about 100 nm thick. When these sections were subjected to silver impregnation, segments of nucleolonema were visualized. Most of them were found to contain achromatic holes. These holes apparently corresponded to the fibrillar centres seen with the electron microscope. According to the profiles of the holes, nucleolonema structures were classified into three types: (1) nucleolonema with no distinct holes, (2) those with beaded holes, and (3) those with cylindrical holes. The thicknesses were 0.7–0.8, 0.9–1.2, or 1.2–1.4 μm for nucleolonemata with no holes, beaded holes, or cylindrical holes, respectively. The argyrophilic wall of nucleolonemata with holes was about 0.4 μm thick, roughly compatible with the thinnest nucleolonema seen in air-dried specimens. The crescent-shaped segments were sometimes observed when the nucleolonema was sectioned transversely, suggesting that the achromatic holes are exposed to the nucleoplasm, in other words, the nucleolonema is partially degraded. Thus, the nucleolonema was not always structurally stable during interphase. The results suggest that the nucleolonemata gradually become knobbly and increase their thickness, with concomitant expansion of the fibrillar centres sometimes degrading into approximately 0.5 μm thick strands.     

Triple staining of normal and degenerating nervous tissue.

A method of counterstaining sections impregnated according to a previously reported modification of the Glees silver impregnation is described. The basis for this counterstain is the Klüver-Barrera luxol fast blue technique. The results are illustrated and the advantages and disadvantages of the procedure are discussed.     

Dichromate-Chlorate Perfusion Prior to Staining Degeneration in Brain and Spinal Cord

A method of fixation compatible with both the Nauta-Gygax and Swank-Davenport procedures for degenerating nerve fibers, which shortens the time required by the former procedure, is as follows: The central nervous system is perfused with a 0.9% aqueous solution of NaCl followed by an aqueous solution containing 5% K₂Cr₂O₇ and 2.5% KClO₃. The central nervous system is then hardened in 10% formalin for 1-3 days. Tissue for Marchi-type staining can be taken at this stage. For silver staining, the processing is continued by immersion overnight in 10% formalin in 20% alcohol, and frozen sections cut the next day. Sections, up to 50μ in thickness, are collected in 10% formalin and impregnated by the Nauta-Gygax technique. Best results are obtained by impregnating within 24-48 hr after sectioning.