Application of the Periodic Acid-Schiff Procedure to Tissue Blocks

The periodic acid-Schiff procedure can be used for staining en bloc by incorporating the periodic acid with the fixing fluid. After simultaneous fixation and oxidation for 48 hr at room temperature and subsequent staining in Schiff reagent the tissues are dehydrated, embedded in paraffin and sectioned. Of two fixatives used, 95% alcohol proved superior to 10% formalin. Various concentrations of periodic acid (0.1-2.0%) yielded equally good results, thus the use of the lower concentrations is feasible and preferable. Fixation and oxidation simultaneously or separately yielded equally satisfactory results and in view of the time saved in the simultaneous method the authors recommend it. Using similar time of fixation and oxidation, satisfactory results were obtained with the intestine of rat after 3 hr of exposure to Schiff reagent. A longer period of exposure (up to 48 hr) was needed for comparable results with the kidney and liver.     

Gross Demonstration of the Mammalian Atrioventricular Bundle by A Periodic Acid-Schiff Procedure

The following procedure stains the atrioventricular conduction system selectively. (1) Wash the fresh heart with physiological saline solution to free it of blood; (2) fix it in 10% formalin containing 0.5% HIO₄ for 1 hr; (3) wash in 3 changes of distilled water for 20 min; (4) keep in 80% alcohol for 12 hr to 2 wk; (5) wash with distilled water; (6) treat with a dilute Schiff's reagent containing 0.1 gm of basic fuchsin per 100 ml for 0.5-2 min; (7) rinse in three changes of 2% Na₂SO₃ in 0.2 N HCI for 3-5 min; (8) wash and examine in 80% alcohol; store in 80% alcohol.     

The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

Luxol Fast Blue Combined With the Periodic Acid-Schiff Procedure for Cytological Staining of Kidney

Rat kidneys fixed in Regaud's fluid were stained by luxol fast blue (LFB), by the periodic acid-Schiff (PAS) method, and by LFB combined with PAS. When used separately the PAS stains the brush border, hyaline droplets and basement membranes reddish, the LFB stains the mitochondria, hyaline bodies and basement membranes greenish-blue. The combined LFB-PAS method stains the brush border reddish and the mitochondria dark blue, while the hyaline bodies and basement membranes are purplish colored. The LFB-PAS method provides color contrasts which show cytological features that are particularly significant in the kidney.     

Combined Aldehyde-Fuchsin and Periodic Acid-Schiff Staining of the Pituitary

Aldehyde-fuchsin and the periodic acid-Schiff procedure can be applied in sequence to the same tissue section and combined with a stain for acidophils, such as orange G, for routine analysis of the pituitary. This is made possible by fixation in a mixture containing 5% chrome-alum, 5% HgCl₂ and 5% formalin; control of the pH and the activity of the aldehyde-fuchsin; and increased sensitivity of the Schiff reagent, effected by reducing its SO₂ content. Although designed especially for the pituitary, the procedure is also applicable to other histological problems.     

Application of the Periodic Acid-Schiff Technique to Whole Chick Embryos

The practicability of applying histochemical reactions to bulk staining has been explored by subjecting whole chick embryos at early stages to the periodic acid-Schiff (PAS) reaction. A comparison of the microscopic distribution of PAS positive substances revealed by this procedure with that obtained by the standard routine, i.e., staining of deparaffinized sections on slides, has shown similar localizations of PAS positive material and, in addition, finer morphological detail and more intensive reactions by staining the specimens in toto. The following method is recommended for chick embryos between stages 11-17 (Hamburger and Hamilton): Fixation in Gendre's fluid at 4°C; oxidation with alcoholic buffered periodic acid, 15 min; rinsing in distilled water, 10 min; Schift's reagent, 30 min; 3 sulfite rinses, 5 min each; running tap water, 10 min; dehydration, clearing and double-embedding in celloidin and paraffin.     

Demonstration of Alkaline Phosphatase and Periodic Acid-Schiff Positive Material in the Same Section

Alkaline phosphatase activity and periodic acid-Schiff (PAS) positive material were demonstrated in the same section by producing a gold-toned silver deposit at the sites of enzyme activity, followed by the PAS reaction. The glycerophosphate incubating mixture was modified by substituting AgNO₃ (final concentration about 0.17%) for the magnesium salt, and, after a slightly lengthened incubation, the silver was reduced by means of a fine grain photographic developer. Gold toning was followed by the regular PAS staining procedure. Gold deposits in the sites of enzyme activity were fully resistant to the action of periodic acid.     

Basic Dye Counterstaining of Sections Impregnated by the Golgi-Cox Method

Celloidin blocks of Golgi-Cox impregnated material are cut at 50 μ, the sections collected in 70% alcohol, transferred to a 3:1 mixture of absolute alcohol and chloroform for 2 min, and then stored in xylene or toluene for at least 3 min, or up to 2 wk until processed further. Mounting is done on glass slides which have been coated with fresh egg albumen diluted in 0.2% ammonia water (or a 0.5% solution of dry powdered egg albumen) and then dried at 60°C overnight. For attachment to these coated slides, sections are first soaked for 2-3 min in a freshly prepared mixture of methyl benzoate, 50 ml; benzyl alcohol, 200 ml; chloroform, 150 ml; and then transferred quickly to the slides by means of a brush. After 2-3 min the chloroform evaporates and the celloidin softens. The slides are then immersed in toluene which hardens the celloidin and anchors the sections to the slides. Alcohols of descending concentrations to 40% are followed by alkalinizations, first in: absolute alcohol, 40 ml; strong ammonia water 60 ml, for 2 min, then in: absolute alcohol, 70 ml; strong ammonia water, 30 ml, for 1 hr. Excess alkali is then removed by 70% and 40% alcohol, 2 min each, and a 10 min wash in running tap water. Bleaching in 1% Na₂S₂O₃, for 10 min and washing again in tap water for 10 min completes the process preliminary to staining. The preparations are then stained for 90 min in an aqueous solution of either 0.5% cresylecht violet, neutral red, or Darrow red, buffered at pH 3.6. Dehydration and differentiation in ascending grades of alcohol, clearing with toluene or xylene, and applying a cover glass with a mounting medium having a refractive index of about 1.61 completes the process.     

Adaptation of the Millon, Sudan Black B, and Periodic Acid-Schiff Technics for Block Staining of Insect Tissues

Spermatophores and reproductive systems of the beetle, Lytta nuttalli Say, fixed in Bouin's aqueous picroformol or buffered 10% neutral formol were stained in toto by the Millon, Sudan black B and periodic acid-Schiff reactions as follows. Millon: after excess fixative is removed in 70% ethanol, specimens are brought to water, stained in Millon's reagent at 60 C for 1 hr, rinsed in 2% aqueous nitric acid at 40-50 C, dehydrated rapidly, cleared, embedded and sectioned as usual. Sudan black B: specimens are taken to absolute ethanol, stained in a saturated solution of Sudan black B in absolute ethanol at room temperature for 24-48 hr, rinsed and cleared in xylene, embedded and sectioned. PAS: specimens are brought to water, oxidized in 0.5 aqueous HIO₄ at 37 C for 30 min, washed in 2 changes of water, stained in Schiif reagent at room temperature for 1 hr, rinsed in 3 changes of 0.5% aqueous potassium metabisulfite, washed in running water for 10-15 min, dehydrated, cleared, embedded and sectioned. All 3 methods produced their characteristic staining in specimens up to 3 mm thick     

Differential Staining of Mucin Granules from Epoxy Resin Sections by a Phosphotungstic Acid-Methyl Green Procedure

After treatment of epoxy resin semithin sections from glutaraldehyde fixed rat large intestine with 5% aqueous phosphotungstic acid (PTA), staining with unpurified 0.2% solutions of methyl green at 60 C for 5 min produces a color differentiation between mucin granules of goblet cells. Some mucin granules and the glycocalyx appear deep green while the remaining granules, luminal mucin and collagen fibers are pink. The known contamination of unpurified methyl green with crystal violet seems to be responsible for the pink staining reaction of the latter structures, which also present an orange-red fluorescence under green exciting light. Electron microscopic observations show selective contrast of mucin granules which appear with a different amount of PTA deposits. This procedure is useful to reveal the heterogeneity of mucin granules in light and electron microscopy.     

Detection of a Fine Lamellar Gridwork After Degradation of Ocular Melanin Granules by Cultured Peritoneal Macrophages

In the present study we investigated by electron microscopy whether melanin granules derived from choroidal melanocytes and retinal pigment epithelium of cattle could be degraded in the phagolysosomes of cultured murine macrophages. It was found that degradation of ocular melanin is possible by the lysosomes of these macrophages. During degradation of the melanin granules an internal gridwork of fine concentric, highly ordered membranes, 3-4 nm thick, became visible. These membranes may represent remnants of the melanin polymer in the original melanosome or may result from self-assembly of degradation products. Early-stage melanosome-like structures also appeared during digestion of these melanin granules. Melanin granules that seemed to break down into smaller fragments without any visible internal structure were also observed.     

The Increased Basophilia of Tissue Proteins After Oxidation with Periodic Acid

Oxidation by periodic acid (a 1% aqueous solution for 1 hour at 37°C.) greatly enhances the basophilic staining normally present in the keratinized portions of epidermis and hair. It also induces strong basophilia in purified fibrin and in the cytoplasm of smooth and striated muscle fibers, endothelium and the cells of the pancreatic islets and acini Proof is offered that this observed basophilia differs from that of either acid mucopolysaccharides or nucleoproteins. The conclusion is reached that, besides revealing polysaccharides occurring in tissue sections, periodic acid also brings about the formation of strongly acid, basophilic groups. These acid groups have not been identified, but since they may be formed most readily in regions of high sulfur content, it is suggested that sulfide and sulfhydryl groups may be oxidized to the corresponding sulfonic acids which could account for the observed characteristics of the basophilic substances.     

Estimation of Cytotoxic Injury to Gastric Parietal Cells by Trypan Blue Exclusion Test Followed by Hematoxylin-Eosin Counterstaining of Fixed Smears

A modification of a trypan blue exclusion technique for detection of cyto-toxicity is described with the use of a trypan blue exclusion test followed in sequence by hematoxylin-eosin counterstaining of smears permanently fixed. This technique, as applied to the suspension of parietal cells, permits the distinction between intact, moderately damaged, and severely damaged cells, determination of their ratio to each other, and an insight into the interrelations of aggressive cells (sensitized lymphocytes) to target cells (parietal cells) in the permanently fixed gastric cells suspension.     

神经干细胞脑内移植后的存活、迁移和分化

从胚胎或成体大鼠脑组织、人胚脑组织均能分离到神经干细胞 ,将它们进行体外原代培养扩增或永生化后植入脑内 ,均能观察到其在脑内的迁移和分化现象。其分化能力主要取决于移植部位的脑内微环境 ,但这种影响作用是相对的。同时 ,体外培养环境如培养时间和细胞融合程度、维甲酸类诱导分化剂处理、NGF转导处理再移植或与嗜铬细胞 (分泌NGF)共移植等 ,也能决定神经干细胞脑内移植后向神经元方向分化的能力。神经干细胞移植为中枢神经系统功能重建和神经再生带来新的希望。     

Advantages in the Use of Aldehyde Fuchsin with Van Gieson's Picro-Fuchsin Counterstaining for Differentiating Bone and Cartilage

Specimens of bone were fixed in 10% neutral phosphate-buffered formalin or in Bouin's fluid and decalcified in 10% formic acid buffered with 10% sodium citrate. Materials were embedded in paraffin and 4-5 μ sections attached to slides were oxidized with 0.5% KMnO₄, decolorized in 1% oxalic acid, stained with aldehyde fuchsin, and counter-stained with Van Gieson's picro-fuchsin. Sections were dehydrated, cleared and mounted in a synthetic resin. Microscopically, the differentiation between bone and cartilage was seen as red and purple respectively, with connective tissue red; muscle and erythrocytes, yellow; and elastic fibres purple. The areas occupied by bone, cartilage and erythrocytes could be compared, and also the depth to which cartilage extended into the ossified sites. The advantages of this staining combination are: good contrasts in colour, ease of applying the stain, and virtual self-differentiation of the staining solutions.     

Degradation of Native Starch Granules by Barley alpha-Glucosidases

The initial hydrolysis of native (unboiled) starch granules in germinating cereal kernels is considered to be due to α-amylases. We report that barley (Hordeum vulgare L.) seed α-glucosidases (EC 3.2.1.20) can hydrolyze native starch granules isolated from barley kernels and can do so at rates comparable to those of the predominant α-amylase isozymes. Two α-glucosidase charge isoforms were used individually and in combination with purified barley α-amylases to study in vitro starch digestion. Dramatic synergism, as much as 10.7-fold, of native starch granule hydrolysis, as determined by reducing sugar production, occurred when high pl α-glucosidase was combined with either high or low pl α-amylase. Synergism was also found when low pl α-glucosidase was combined with α-amylases. Scanning electron micrographs revealed that starch granule degradation by α-amylases alone occurred specifically at the equatorial grooves of lenticular granules. Granules hydrolyzed by combinations of α-glucosidases and α-amylases exhibited larger and more numerous holes on granule surfaces than did those granules attacked by α-amylase alone. As the presence of α-glucosidases resulted in more areas being susceptible to hydrolysis, we propose that this synergism is due, in part, to the ability of the α-glucosidases to hydrolyze glucosidic bonds other than α-1,4- and α-1,6- that are present at the granule surface, thereby eliminating bonds which were barriers to hydrolysis by α-amylases. Since both α-glucosidase and α-amylase are synthesized in aleurone cells during germination and secreted to the endosperm, the synergism documented here may function in vivo as well as in vitro.     

Control of Differentiation by GTP

Abstract

A partial deficienc of GTP or GDP induces the differentiation of microorganisms and certain cells of higher organisms. The mechanism of this control may be retained during evolution.