Histochemical Use Of Lead Tetra-Acetate I. Cleavage Of 1-2 Glycols

Lead tetra-acetate acts specifically to split the carbon-carbon single bond of the 1,2-glycol linkage to produce aldehyde radicals which may then be demonstrated by means of leucofuchsin, 2,4-dinitrophenlyhydrazine, or p-nitrophenylhydrazine. Routinely prepared slide sections from tissues fixed in 10% formalin are run down to 95% alcohol, rinsed in glacial acetic acid and then treated for 2 minutes in a saturated solution of lead tetra-acetate in glacial acetic acid with 5 g. of potassium acetate added for each 100 ml. of reagent. The sections are then washed in distilled water and placed in leucofuchsin for 10 minutes, or in a saturated 30% alcoholic solution of p-nitrophenylhydrazine for 5 minutes or 2,4-dini-trophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 minutes if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofuchsin or yellow by the nitrophenylhydrazines.     

Histochemical Use of Sodium Bismuthate

Histochemical 1,2-glycoI cleavage, similar to that obtained with periodic acid and lead tetraacetate, may be obtained with sodium bismuthate. Routinely prepared slide sections, from tissues fixed in 10% formalin, are run down through xylene and graded alcohols to water and then oxidized for three minutes in a 1% sodium bismuthate 20% aqueous phosphoric acid solution. The oxidizing solution must be freshly prepared and used immediately. Following oxidation, sections are rinsed 15 sec. in IN HC1 to remove bismuth pentoxide precipitate, a by-product of the reaction. The sections are then washed in distilled water and placed in leuco-fushsin for 10 min., or in a saturated 30%) alcoholic solution of p-nitrophenylhydrazine for 5 min. or 2,4-dinitrophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 min. if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofushsin or yellow by the nitrophenylhydrazines.     

Histochemical Use of Sodium Bismuthate

Histochemical 1,2-glycoI cleavage, similar to that obtained with periodic acid and lead tetraacetate, may be obtained with sodium bismuthate. Routinely prepared slide sections, from tissues fixed in 10% formalin, are run down through xylene and graded alcohols to water and then oxidized for three minutes in a 1% sodium bismuthate 20% aqueous phosphoric acid solution. The oxidizing solution must be freshly prepared and used immediately. Following oxidation, sections are rinsed 15 sec. in IN HC1 to remove bismuth pentoxide precipitate, a by-product of the reaction. The sections are then washed in distilled water and placed in leuco-fushsin for 10 min., or in a saturated 30%) alcoholic solution of p-nitrophenylhydrazine for 5 min. or 2,4-dinitrophenylhydrazine for 30 minutes. After staining, the sections are rinsed in 30% alcohol if the nitrophenylhydrazines were used, or in the standard dilute sulfite bath followed by running tap water for 5 min. if leucofuchsin were used. Sections are routinely dehydrated, cleared, and covered. On examination, the sites of 1,2-glycol linkages will be stained violet by leucofushsin or yellow by the nitrophenylhydrazines.     

Histochemical Demonstration of Carbonic Anhydrase Activity

Fresh tissue slices fixed in chilled acetone for 1 hour and washed in distilled water for 10-30 minutes were incubated for 30-45 minutes at 37°C. in the freshly prepared incubating mixture: filtrate of a mixture of 8% sodium bicarbonate, 100 ml., and MnCl₂·4H₂O, 1 g. After washing in distilled water for 1 hour, they were dehydrated and embedded in paraffin. Sections were cut 15-20μU, deparaffinized, rinsed in absolute alcohol and placed in a 0.1% solution of potassium periodate for 48 hours at 37°C. The mounted sections were counterstained (if desired), dehydrated in alcohol, cleared in xylene (not carbol-xylene) and mounted in balsam. Many brown granules were produced on the sites of enzyme activity by this procedure. The results obtained seem to be in good agreement with previous findings by biochemical determinations.     

Suppression of Connective Tissue Impregnation in a Silver Technique for Demonstrating Nerve Fibers

A tissue pretreatment technique is introduced which effectively suppresses the silver impregnation of connective tissue and nompecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcobol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginnins with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilidng fresh hrozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation H suppressed by the use of mercuric chloride in the fixative and that the background supprgsion is related to the short fixation time with formol-sublimate.     

Suppression of connective tissue impregnation in a silver technique for demonstrating nerve fibers.

A tissue pretreatment is introduced which effectively suppresses the silver impregnation of connective tissue and nonspecific background elements in peripheral nerve. The result is a selective impregnation of nerve fibers. The procedure utilizes fresh frozen sections and can be used with the Holmes (1947) or Bodian (1936) techniques. Fresh frozen sections are cut at 10 microns, mounted on slides and air dried for 5 minutes. They are fixed for 30 minutes in formol-sublimate (10% formalin saturated with mercuric chloride) and then placed into 0.5% iodine in 70% alcohol for 5 minutes followed by bleaching in 2.5% sodium thiosulfate for 2 minutes. After washing in running tap water for 10 minutes and a brief rinse in distilled water, impregnation is accomplished by the Holmes (1947) or Bodian (1936) procedure beginning with the step containing the aqueous silver solution. The results show an absence of impregnation of connective tissue and nonspecific background. The technique is simple, rapid, and, by utilizing fresh frozen sections, can be used for other histological and histochemical purposes. Several experiments were done to determine the causes of the connective tissue and background suppression. The air drying step was omitted; the sections were fixed in formalin without mercuric chloride; and the formol-sublimate fixation time was increased. The results suggest that connective tissue impregnation is suppressed by the use of mercuric chloride in the fixative and that the background suppression is related to the short fixation time with formolsublimate.     

Biological Stain Commission Membership,June 1959

Frozen sections of avian tissue fixed 7 days or longer in 10% formalin or formol-saline are cut at 20-50 μ, left in distilled water for 2 hr, and placed in 0.002% aqueous AgNO₃ for 3-4 days. Subsequent procedure is essentially that of Weddell and Glees. Sections are placed in 20% AgNO₃ for 30 min, then carried through 3 baths of 3% formalin in less than 10 min. Immediately thereafter they are washed 1-2 sec in a 0.1% solution of NH₄OH (cone) and placed in the ammoniacal silver solution (made with 20% AgNO₃) until the nerves become distinct, as seen under a microscope; usually, in about 15 min. After washing briefly, the sections are fixed in 5% Na₂S₂O₃ for 3-10 min, dehydrated, cleared, and mounted in the usual way.     

Hydrogen sulfide alleviates uranium‐induced rat hepatocyte cytotoxicity via inhibiting Nox4/ROS/p38 MAPK pathway

As a gasotransmitter, hydrogen sulfide (H₂S) plays a crucial role in regulating the signaling pathway mediated by oxidative stress. The purpose of this study was to investigate the protective effects of H ₂S on uranium‐induced rat hepatocyte cytotoxicity. Primary hepatocytes were isolated and cultured from Sprague Dawley rat liver tissues. After pretreating with sodium hydrosulfide (an H ₂S donor) for 1 hour (or GKT‐136901 for 30 minutes), hepatocytes were treated by uranyl acetate for 24 hours. Cell viability, reactive oxygen species (ROS), malondialdehyde (MDA), NADPH oxidase 4 (Nox4), and p38 mitogen‐activated protein kinase (p38 MAPK) phosphorylation were respectively determined. The effects of direct inhibition of Nox4 expression by GKT‐136901 (a Nox4 inhibitor) on ROS and phospho‐p38 MAPK levels were examined in uranium‐treated hepatocytes. The results implicate that H ₂S can afford protection of rat hepatocytes against uranium‐induced adverse effects through attenuating oxidative stress via prohibiting Nox4/ROS/p38 MAPK signaling.     

PREFERENTIAL STAINING OF NUCLEIC ACID-CONTAINING STRUCTURES FOR ELECTRON MICROSCOPY

Oriented fibres of extracted nucleohistone were employed as test material in a study of satisfactory fixation, embedding, and staining methods for structures containing a high proportion of nucleic acid. Fixation in buffered osmium tetroxide solution at pH 6, containing 10^-2 M Ca⁺⁺, and embedding in Araldite enabled sections of the fibres to be cut in which the orientation was well preserved. These could be strongly stained in 2 per cent aqueous uranyl acetate, and showed considerable fine structure. Certain regions in the nuclei of whole thymus tissue could also be strongly stained by the same procedure, and were identical with the regions stained by the Feulgen procedure in adjacent sections. Moreover, purified DNA was found to take up almost its own dry weight of uranyl acetate from 2 per cent aqueous solution. Strongest staining of whole tissue was obtained with very short fixation times-5 minutes or so at 0°C. Particularly intense staining was obtained when such tissue stained in uranyl acetate was further stained with lead hydroxide. Although the patterns of staining by lead hydroxide alone and by uranyl acetate were similar in tissues fixed for longer times (½ hour to 2 hours, at 0°C or 20°C), in briefly fixed material the DNA-containing regions appeared relatively unstained by lead hydroxide alone, whilst often there was appreciable staining of RNA-containing structures. Observations on the staining of some viruses by similar techniques are also described.     

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.     

Sectioning and Staining Whole Heads of Small Animals

Serial sections showing minimal disruption in delicate structures of the heads of adult small vertebrates were produced with regularity when the heads were: (1) fixed in Bouin's picro-formol; (2) decalcified electrolytically to a radiographically-determined end point; (3) dehydrated in alcohols to 95%; (4) cleared in iso-amyl acetate; (5) infiltrated with concentrations (to 40%) of low viscosity nitrocellulose in iso-amyl acetate; (6) embedded in the same material by hardening it with chloroform; and (7) soaked in a mixture of 95% ethyl alcohol and glycerol (3:1) prior to sectioning on a sliding microtome. Following affixation to slides, the nitrocellulose matrix was dissolved from the sections and they were hydrated. Thin sections stained well with a standard Mallory technique. Thick sections were treated with the same stain, but good results depended upon empirical determination of times to be used in the procedure, and the introduction of water and 95% alcohol extraction-differentiation steps for red-staining and blue-staining tissues, respectively.     

Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues

For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to decalcify the tissues. We evaluated the usefulness of various acid-based decalcifying reagents with reference to 28 S rRNA staining by in situ hybridization using a thymine-thymine dimerized oligonucleotide probe. The reagents evaluated were 10% nitric acid, 10% HCl, 5% formic acid, 5% trichloroacetic acid, Morse’s solution, Plank-Rychlo’s solution, and K-CX solution, all of which are commonly used to decalcify tissues, and their effects on retention of morphology and RNA were compared with EDTA-based solutions. When normal mouse mandible was used as a model tissue, well-preserved morphology of ameloblasts was obtained from sections decalcified with Morse’s solution, 10% HCl, Plank-Rychlo’s solution, and K-CX solution, and best retention of 28 S rRNA was obtained with 5% formic acid and Morse’s solution. We recommend Morse’s solution to decalcify tissues to be processed for the rapid analysis of specific RNA expression. Indeed, we detected specific mRNAs strongly in sections treated with Morse’s solution, and quantitative analysis showed that the ratio of signal intensities of 28 S rRNA and the specific mRNAs correlated with each other depending on decalcifying solutions. Accepted: 3 January 2000     

Hydrogen Sulfide Improves the Vase Life and Quality of Cut Roses and Chrysanthemums

Recent studies indicate that hydrogen sulfide (H₂S) plays various physiological roles in plants. However, whether H₂S participates in the postharvest senescence in cut flowers remains unknown. In this study, the regulatory roles of H₂S during the senescence of cut roses (Rosa hybrida L.) and chrysanthemums (Dendranthema morifolium Ramat.) were investigated. The results showed that compared with the control (distilled water), the 50 μM sodium hydrosulfide (NaHS) treatment, a H₂S donor, extended the vase life of cut roses to 9.3 days and their flower diameter also showed an increment of 22.7% after 4 days treatment. Treatments with 30 μM NaHS significantly prolonged the vase life of cut chrysanthemums to 8.87 days and the flower diameter was 13.21% longer than the control on day 6. Additionally, results also indicated that a 30 or 50 μM NaHS treatment effectively decreased the rate of fresh weight changes and O²⁻ production and H₂O₂ content, increased the levels of soluble sugar, soluble protein, anthocyanin and carotenoid, and enhanced the activities of antioxidant enzymes (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase) of cut roses and chrysanthemums in comparison with the control, implying that H₂S might be involved in regulating the osmotic balance, antioxidant system and the degradation of nutrient and pigments. Altogether, H₂S at proper doses might play an important role in improving the longevity and quality of cut roses and chrysanthemums by maintaining water balance, reducing the degradation of pigments and nutrient and enhancing antioxidant capacity.

     

Differentiation of Articular and Epiphysial Cartilage, Based on Resorcin-CrystaeL Violet and Picro-Fuchsin Staining of Chondroitin Sulphate and Keratosulphate

Formalin-fixed, decalcified knee joints of young vertebrates were embedded in paraffin wax and cut at 4 μ. Sections were stained in Harris' Haematoxylin, washed in tap water, then immersed in the following staining solution for 60 min: crystal violet, 1 gm; resorcin, 2 gm; distilled water, 100 ml; boiled for 3 min, with constant stirring. After adding 30 ml of 30% FeCl₃, it was boiled for 3 min more. The solution was filtered. The precipitate was washed oil with 50 ml of distilled water and 100 ml of absolute alcohol added. This was combined with the original filtrate and boiled for 5 min. The solution was filtered once more, the precipitate discarded and 2 ml of cone. HC1 added. After cooling, the solution was ready for use. Sections were then washed briefly in tap water, stained in van Gieson's picro-fuchsin for 2 min, and differentiated as they were dehydrated and brought to Xylene. The sections were mounted in a synthetic resin (D.P.X.). Articular type cartilage stains red and growth cartilage blue.     

A technique for retrieving antigens in formalin-fixed, routinely acid-decalcified, celloidin-embedded human temporal bone sections for immunohistochemistry.

The application of immunohistochemistry to routinely decalcified, celloidin-embedded human temporal bone sections has been hampered because of antigen loss during processing of the specimens. To our knowledge, there has been no published report to date describing immunohistochemical staining of such tissues suitable for examination by light microscopy. Here we report a novel antigen retrieval technique which can be successfully used to stain a variety of antigens in routinely formalin-fixed, trichloroacetic acid-decalcified, celloidin-embedded human temporal bone sections. The new procedure reported here for decalcified human temporal bone tissues simply requires immersing slides for 30 min at room temperature in an antigen retrieval solution. A total of 60 decalcified, celloidin-embedded human temporal bone tissues were tested with monoclonal antibodies (MAb) to 15 different antigens. Of these, 12 MAb showed definite positive staining, while three were negative. This technique may prove very useful in studying the expression of various antigens by immunohistochemistry in formalin-fixed, acid-decalcified, celloidin-embedded tissues.     

Synaptology of the claustrum in the rat

A great deal of information is available on the morphology of the claustrum in various animal species, as well as on its neuronal distribution and relationships with the cerebral cortex and other nuclei. However, no research has been performed on synaptic organization. Here we report an ultrastructural study performed on 7 male albino rats of the Wistar strain weighing 270-310 g. Five rats were sacrificed by prolonging general anesthesia with diethyl ether until death. Three of these rats were secured to the stereotaxic atlas coordinates of Paxinos and Watson (1982); the claustrum area was marked by injecting 1 microliter of a 10% Evans Blue solution into the nucleus. The brain was then removed from the skull, cut into 2-3 mm thick coronal sections, and tissue samples taken from the area immediately adjacent to the marked area were immersed in 2% OsO4 buffered with 2% potassium dichromate containing 0.2% CaCl2 at pH 7.7 (Gobel, 1968). After dehydration they were embedded in Durcupan and the ultrafine sections were stained with uranyl acetate and lead citrate and observed with either a Zeiss 9S2 or a Hitachi H 800 electron microscope. The samples from two other rats, taken with the stereotaxic techniques described, were fixed for 12 h in 0.6 potassium permanganate solution buffered with veronal-acetate at pH 7.4 (Luft, 1956). After processing for electron microscopy, a portion of the sections were used without any contrast medium and the remainder were stained with uranyl acetate and lead citrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructure of cardiac muscle of Trochosa terricola Thor., Pardosa amentata Clerck,P. pullata Clerck,and Pisaura mirabilis Clerck (Araneae: Lycosidae,Pisauridae)

Summary The ultrastructure of the cardiac muscle in some araneae has been investigated. The sarcolemma invaginates at the Z band and may extend into the cells through several myofibrils. Numerous T-tubules are given off both from sarcolemmal invaginations and also directly from the cell surface. In passing through the Z band, the luminal diameter of T-tubules greatly increases. Dyadic and a few triadic couplings are found mainly at the A-I level. Peripheral couplings were not seen.The ruthenium red solutions employed were prepared as described by Myklebust (1975), but the fixative contained 2% sucrose. The hearts were fixed for 3 1/2h in the ice cold solution, washed in buffer and post-fixed for 1 1/2h in a ruthenium red/OsO₄ solution. Dehydration and embedding was performed as described above. Sections were cut on a Reichert Om U2 microtome, stained with lead citrate (Reynolds, 1963) and examined in a Philips 300 electron microscope at 80 kV.Semithin sections for light microscopy were stained with toluidine blue (Mercer and Birbeck, 1972).I am much obliged to cand. real Erling Hauge for identifying the specimens used in the investigation proportion of relaxed sarcomeres. The fixed pieces were washed in a buffered sucrose solution and post-fixed in a 1% OsO₄ solution buffered with sodium cacodylate for 1 h. Following washes in a compatible buffer and in distilled water, the tissue was stained en bloc for l h in a 2% aqueous uranyl acetate solution. The tissue was dehydrated through cold acetone and embedded in Epon 812.     

A Buffered Giemsa-Bodian Stain for Neurological Material

A buffered Giemsa counterstain for the Bodian method is described. It is very useful for bringing out Nissl substance and nerve fibers in the same section. Bouin perfused or formalin fixed material from mammals, amphibia, reptiles and fish was used. After fixation, all tissues were decalcified for at least a week in 50% formic acid (1 part) and 20% sodium citrate (1 part). This was washed out thoroughly. The method described by Bodian (1936) was followed except for the following minor changes: Winthrop Protargol (Strong Protein Silver) Batch N346BJ was used exclusively; all glassware was cleaned with acid prior to setting up the stain; before developing, the sections were washed in warm tap water for 10 minutes; the gold chloride was chilled before use. The sections were then put into buffered Giemsa for 24 hours. Stock Giemsa: 0.75 grams powdered Giemsa (Coleman and Bell, Certification No. CGe-3) was dissolved in 50 ml. of glycerin overnight in the oven, then 50 ml. of methyl absolute alcohol were added. To 3 ml. of Giemsa stock solution, 87 ml. of distilled water buffered to pH 5.3 with 10 ml. of Sorensen's buffers were added and the solution filtered. Coleman buffer tablets gave best results at pH 5.0. Sections were then rinsed in 95% alcohol, two changes of absolute alcohol, two changes of xylene, and were then mounted in Clarite.     

A Golgi-Electron Microscope Method for Insect Nervous Tissue

Golgi's light microscopic method of selective silver impregnation for nervous tissue combined with electron microscopy appears to offer a promising method for working out the detailed anatomy of individual neurons and their connections. Insect nervous tissue is fixed in a mixture of 2% paraformaldehyde and 21/2% glutaraldehyde in Millonig's buffer (pH 7.2) before postfixation for 12 hours in a solution brought to pH 7.2 with KOH containing 2% potassium dichromate, 1% osmium tetroxide and 2% D-glucose. The tissue is then transferred to a solution of 4% potassium dichromate for 1 day; and for 1-2 days to a 0.75% silver nitrate solution. After dehydration and embedding in Araldite, 50μm sections am made. Areas of interest are cut from these sections and re-embedded in silicone molds. Ultrathin sections are then cut and stained with uranyl acetate and lead citrate. The Golgi method described here gives good results at the level of both light and electron microscopy.     

Tissue Shrinkage Caused by Attachment of Paraffin Sections to Slides: its Effects on Staining

Sections were cut from a wide variety of tissues, and those from each block were divided into four groups before attaching and drying on slides. Four commonly accepted sources of heat were used for drying: (a) gas hotplate set at 65° C; (b) incubator, 37°; (c) oven, 56°; and (d) room temperature, 20°. After drying, the sections were stained, then examined for intensity of staining and for distortion caused by shrinkage. With both soft and decalcified tissue stained by haematoxylin and eosin, the best results occurred in the sections dried at 20° C; the next best at 37°. When stained by Van Gieson's method, both types of tissues were best after 20° drying, but the second-best group showed differences in favour of 56° for soft tissues and 37° for decalcified. After drying decalcified tissue at 65°, the staining of collagen by acid fuchsin was almost completely absent. When impregnated with silver, for reticulin, the best results for soft tissues were after 56° drying; second best, 20°; but decalcified tissues showed a reversal of this order. After PAS, there was an increasing intensity of staining from 20° to 65°, with soft tissue; evidence that histochemical interpretation could be strongly influenced by drying temperature.