A Tri-Basic-Dye Stain for Nerve Cells

A new staining method has been developed for the study of nerve cells and Nissl granules which combines three basic dyes, cresylecht violet, toluidine blue and thionin. The use of this tri-basic-dye stain results in finished preparations that are critically stained and permanent. Paraffin sections (4 μ sections preferably) are mounted on slides by the starch medium, deparaffinized and stained by the tribasic staining solution. After differentiation in acidified distilled water, sections are dehydrated, returned to stain solution and again dehydrated, then cleared and mounted in Clarite. Various vertebrate material including normal and pathological human tissues have been stained with this triple dye solution. Especially for pathological material, re-immersion of slides in the staining and 80% alcohol solutions before mounting, differentially intensifies the staining reaction. Fixatives used were 10% formalin, 95% alcohol, Bouin and formalin-Bouin (10% formalin followed by Bouin).     

Fixative-Stain Sequences for Selective Demonstration of Juxtaglomerular Cells

The effects of 31 fixatives, containing alcohol, acids, formalin and metallic salts, and representing many of the standard fixatives, were observed for selectivity and intensity of staining of juxtaglomerular granules in mouse kidney. Four staining methods: 1:400,000 aqueous methyl violet 2B; Bowie's ethyl violet-Biebrich scarlet; 1:200,000 aldehyde fuchsin; and periodic acid-Schiff were used. Fixatives containing HgCl₂, trichloroacetic acid or formalin were found to be the most satisfactory for subsequent staining of the granules.     

Studies on Bovine Mast Cells: I. The Effect of Formalin Fixation

Studies are reported on the effect of aqueous formalin fixation on the bovine mast cell. Paraffin sections of bovine skin were prepared from tissues fixed for 7 to 72 hr in 10% neutral formalin in saline, and from tissues which were treated with water for various times both before and after formalin fixation. Metachromatic halos, scattering of the granules, and fraying of the cell outline, were seen in the mast cells of tissues washed in water before fixation. Exposure to water after fixation did not produce these artifacts. The tendency towards orthochromatic staining, and the occurrence of perinuclear clear zones are probably effects of the formalin and not of overstaining or of exposure of the tissue to water. The majority of the metachromatic material in the bovine mast cell is water soluble, and may be removed by washing in water before fixation, whereas the granules are relatively resistant to water. The optimum time of formalin fixation of bovine skin to permit studies on the metachromatic material of the mast cells was 24 to 36 hr.     

Preparing Plant Root Tip Cells for Microscopical Examination

The technics generally used for preparing root tip cells for microscopical examination destroy mitochondria and other cytoplasmic particles and remove lipoidal material. Fixation in a bichromate solution followed by treatment with osmic acid preserves these granules through normal embedding procedures (cf. Zirkle, 1929; Newcomer, 1940); also fixation in neutral formalin and embedding in the water-soluble wax, Aquax, may completely preserve lipoidal matter, and thus the mitochondria. The separation of cells in squash preparations usually entails acid hydrolysis of the intercellular cement. Treatment for one hour with a 5% solution of a commercial pectinase powder in a 1% aqueous solution of peptone allows good separation of cells of bean root tips fixed in acetic-alcohol. By this method it has been demonstrated that the Feulgen hydrolysis removes cytoplasmic and nucleolar RNA. To preserve the mitochondria it is advisable to immerse the bean roots in a 5% aqueous solution of polyvinyl alcohol for 24 hours and to separate the cells with a 10% solution of pectinase in a 1% peptone solution. This procedure preserves the granules, leaves the nucleus optically homogeneous, and gives a result most closely approximating to that observed in living root tip cells.     

The Histological Examination of Mummified Material

Tissue from Egyptian mummy material is extremely brittle; hence it was handled in perforated glass tubes during processing. The first (softening) fluid consisted of 96% ethyl alcohol, 30 vol; 1% aqueous formalin, 50 vol; 5% aqueous Na₂CO₃, 20 vol. It was used in a fluid to tissue volume ratio of 100:1 and allowed to act overnight. A special dehydrating sequence: 80% alcohol, 3-6 hr; 8% phenol in 96% alcohol, and absolute alcohol followed by 3 changes of amyl acetate, 6-18 hr each; 3 changes of 1 % celloidin in methyl benzoate, 24 hr each; then through benzene and embedding in paraffin completed the special technic. This allowed regular sectioning and staining to be done successfully.     

Staining Methods For Studying Ingredient Distribution In Baked Cakes

A method is described for preparing cake crumb for sectioning and staining. Previous to embedding, the fat was stained and fixed by exposing small blocks of cake to the fumes from a 5%, freshly-prepared, aqueous solution of osmic acid (OsO₄). This was followed by dehydration in ethyl alcohol and tertiary butyl alcohol, removal of air under vacuum and infiltration with paraffin.

Sections were cut 20 and 9Op thick and mounted with water.

Wax was removed by immersion in xylene. The sections were rehydrated in a series of ethyl alcohol dilutions, from concentrated to dilute, then transferred to distilled water.

Protein was then stained pink by immersion of the slides in an acidified 0.04% water solution of eosin Y, or starch was stained blue with a dilute aqueous solution of iodine. Ten grams iodine and 10 g. KI were dissolved in 25 ml. distilled water. This stock solution was diluted for use one to two hundred times.

The relationship between protein and starch was demonstrated by staining the sections with eosin, differentiating in 50% alcohol and staining with iodine.

When slides of cake crumb were prepared in this way, the fat was stained black, the protein bright pink and the starch granules a dark blue.     

Change of granular and molecular structures of waxy maize and potato starches after treated in alcohols with or without hydrochloric acid

Starches from waxy maize and potato were treated in methanol and 2-propanol either with or without 0.36% hydrochloric acid at 65 °C for 1 h. The granule morphology, molecular structure and pasting properties of the starches were determined and the effects of treatments on the granule and molecular structures of starch were investigated. Starch treated in alcohols without acid showed loss of native order through the hilum of granules, and no obvious molecular degradation was found. However, acid–alcohol treated starch showed many cracks inside granules, and both waxy maize and potato starches showed obvious molecular degradation after treated. Furthermore, the amylose chains and long chains of amylopectin of starch were more easily degraded with acid–alcohol treatment. The pasting viscosity of acid–alcohol treated starches were also obviously less than that of their counterpart native starch and starch after alcohol treatment. The extent of degradation of molecules and the decrease of pasting viscosity on potato starch after acid–alcohol treated were more obvious than that of waxy maize starch. The result indicates that the degradation preferentially occur in the amorphous region when starch treated by acid–alcohol, and the degradation of starch molecules enhances the amorphous excretion and the occurrence of cracks inside the granules.     

Staining of keratin and keratohyalin with the reactive dye levafix red violet E-2BL.

Demonstration of keratin in Zenker-fixed skin and in tissues stored in formalin can be difficult because such material is unsuitable for histochemical studies. A reactive dye, Levafix red violet E-2BL, proved useful for demonstration of keratohyalin and some types of keratin. Formalin-, Zenker- and methacarn-fixed sections were pretreated with alkaline alcohol, stained one hour at 60 C in an aqueous solution containing 0.25% Levafix red violet E-2BL plus 0.25% NaCl, rinsed in buffer solution pH 9, dehydrated and mounted. Keratohyalin granules and stratum corneum were colored red violet; hair and tonofibrils remained unstained. In sections prestained with Mayer's acid hemalum, keratohyalin was dark blue. Sulfonated monoazo dyes without reactive groups colored no tissue structures under the conditions of this technic; apparently, Levafix red violet E-2BL is bound via its reactive group. Polarization microscopic studies suggest binding of Levafix red violet E-2BL by an amorphous matrix of keratin. Correlations with chemical data indicate that the staining patterns parallel the distribution of proteins formed in the stratum granulosum.     

Staining of Keratin and Keratohyalin with the Reactive Dye Levafix Red Violet E-2BL

Demonstration of keratin in Zenker-fired skin and in tissues stored in formalin can be difficult because such material is unsuitable for histochemical studies. A reactive dye, Levafix red violet E-PBL, proved useful for demonstration of keratohyalin and some types of keratin. Formalin-, Zenker- and methacarn-fired sections were pretreated with alkaline alcohol, stained one hour at 60 C in an aqueous solution containing 0.25% Levafix red violet E-2BL plus 0.25% NaC1, rinsed in buffer solution pH 9, dehydrated and mounted. Keratohyalin granules and stratum corneum were colored red violet; hair and tonofibrils remained unstained. In sections prestained with Mayer's acid hemalum, keratohyalin was dark blue. Sulfonated monoazo dyes without reactive groups colored no tissue structures under the conditions of this technic; apparently, Levafix red violet E-2BL is bound via its reactive group. Polarization microscopic studies suggest binding of Levafix red violet E-2BL by an amorphous matrix of keratin. Correlations with chemical data indicate that the staining patterns parallel the distribution of proteins formed in the stratum granulosum.     

Granulation of subtilisin by internal gelation of alginate microspheres for application in detergent formulation

Subtilisin was encapsulated within impact-resistant alginate granules produced by emulsification, internal gelation, and acetone extractive drying. The mechanical and controlled release properties of the granules were modified by adding to the alginate varying levels of formulation excipients, including titanium dioxide, polyvinyl alcohol, microcrystalline cellulose, starch and sucrose. Optimum protease activity and mass yields of 83 and 88%, respectively (mg active subtilisin/g granules), occurred for granules formulated with 3% alginate, 10% starch, 10% titanium dioxide, and 3% subtilisin. Mass losses occurred primarily during the gelation step. Maximum encapsulation efficiency is achieved by using higher molecular weight alginate, increasing the alginate concentration, and carefully controlling process temperature and pH. The strongest granules were obtained at the higher concentrations of medium-G or high-G alginate, while fastest granule dissolution was achieved when a lower concentration of alginate was used in combination with polyvinyl alcohol or microcrystalline cellulose as dispersants. Mechanical properties of alginate granules were found to be unaffected by the different cations employed in matrix gel formation.     

Staining Juxtaglomerular Granules with Basic Fluorescent Stains

Many basic fluorescent dyes stain juxtaglomerular granules to produce characteristic colors in ultraviolet light. The stain is applied to paraffin sections of tissues fixed in 2% calcium acetate-10% formalin or in phosphate-buffered 10% formalin. Procedure: Bring section to water, stain 0.5 min in Delafield hematoxylin, wash in tap water, stain 3 min in a 0.1% aqueous solution of basic fluorescent dye (auramine O, acriflavine, acridine orange, coriphosphine O, acridine yellow, phosphine E, thioflavine T, berberine sulfate, atebrine or rivanol) and differentiate 1 min in 0.1% acetate acid (or omit this step). After washing in tap water, air dry with or without subsequent mounting in a resin. Juxtaglomerular granules stain bright fluorescent yellow or orange against a dark background.     

An Improved Crystal Violet Method with Alkaline Differentiation for Juxtaglomerular Granules

In the previous alkaline crystal violet method for selectively demonstrating juxtaglomerular (JG) granules (Harada 1971), the staining solution was found to be unstable. Subsequent testing has shown that the alkali is equally effective if applied after a nonalkalized aqueous solution of crystal violet has been applied for the staining, thus allowing stable stock solutions of the staining reagents to be used. The new procedure is as follows:

Sections of 4 μ thickness from adult mouse kidney fixed in phosphate-buffered 10% formalin were cut from paraffin-embedded material and attached to slides with albumen adhesive. They were deparaffinized, hydrated, and washed in tap water.     

Enzyme modification of starch granules: formation and retention of cyclomaltodextrins inside starch granules by reaction of cyclomaltodextrin glucanosyltransferase with solid granules

Cyclomaltodextrin glucanosyltransferase (CGTase) was adsorbed into starch granules and allowed to react at 37 degrees C. The reaction was conducted with the granules removed from an aqueous environment, but containing 50% w/w water inside the granule. Reaction for 20 h gave a maximum of 1.4%, w/w of cyclodextrins (CDs) inside the granule. Waxy maize and maize starches gave the highest amounts of CDs (1.3 and 1.4%, respectively), with tapioca and amylomaize-7 starches giving about 50% less (0.9 and 0.6%, respectively). Reaction of a combination of CGTase and isoamylase with solid starch granules gave a 2.6-fold increase in the formation of CDs, with a maximum yield of 3.4 and 100% retention inside waxy maize starch granules.     

A Rapid Method for Preparing Paraffin Serial Sections of Teeth and Their Supporting Structures in the Dog

A method of preparing dogs' teeth for serial sectioning involving the following steps is described. The tissues are fixed in 10% formalin followed by partial dehydration in 80% iso-propyl alcohol; decalcified in 5% nitric acid in 10% formalin; washed for 5-8 hours in running water and neutralized in 5% aqueous sodium sulfate for 24 hours, followed by further washing in running water for another 24 hours. They are then dehydrated over a period of 4 days in increasing grades of iso-propyl alcohol and embedded in tissue-mat in sub-atmospheric pressure of 10 lb. Sections so obtained were better suited for cytological study than celloidin preparations, and serial sections could be obtained in a much shorter time.     

Restoration of the softness and flexibility of cadavers preserved in formalin

Submerging parts of cadavers in a 5% aqueous solution of glacial acetic acid eliminates the undesirable properties of formalin and formalin-containing fixatives such as the irritating and unpleasant smell of formalin and phenol. It also considerably softens the tissues, renders the articulations flexible and makes dissection much easier. No significant bone softening occurs if the process is stopped at a suitable time. Parts of the bodies softened in this way can be kept for years in 70% alcohol without any signs of a loss of flexibility or decomposition.     

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.     

Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II.

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.     

A Modified One-Step Trichrome Stain for Demonstration of Fine Connective Tissue Fibers

Gomori's one-step trichrome procedure was modified to improve coloration of fine connective tissue fibers. Paraffin sections from tissues fixed in alcohol, acetone, Zenkerformol, 10% formalin, Kaiserling's or Carnoy's fluid were mordanted 1 hr at 56 C in Bouin's solution, stained 1 min in a trichrome solution (chromotrope 2R-phosphomolybdic acidaniline blue WS) adjusted to pH 1.3 with HCl, rinsed in 1% aqueous acetic acid, dehydrated and covered. Collagen, reticulum fibers, basement membranes, ring fibers around splenic sinuses, intercalated discs in cardiac muscle and cartilage were colored blue. Nuclei, cytoplasm, fibrin, muscle fibers and elastic fibers were stained red. Pretreatment of sections with Bouin's solution enhanced the affinity of tissues for chromotrope 2R and was found essential for satisfactory coloration of material fixed in alcohol, acetone, formalin or Carnoy's fluid. Because this method does not require differentiation, it gave uniform results even in the hands of inexperienced laboratory trainees. No fading was observed in sections stored for more than 8 yr.     

Histochemical Aspects of the Affinity of Mast Cell Granules for Night Blue

The affinity of mast cell granules for night blue was studied in fresh and fixed rat lip, dog mast cell tumor, normal human ileum, and human mast cell and carcinoid tumors. Fixatives used were 10% formalin, 1% trichloracetic acid in absolute alcohol, and Zenker's and Bouin's fluids. Extractions of fresh tissue with hot water, acids, and bases removed the stainable material or prevented staining, but similar treatment of fixed tissue did not. Hot pyridine was without effect as was chloroform-methanol, but methylation blocked mast cell staining by night blue. Chromic acid oxidation and prolonged Zenker and Bouin fixation also prevented staining. Hyaluronidase treatment was without effect. Sulfhydryl and disulfide linkages were changed without altering the stainability.     

ULTRASTRUCTURAL LOCALIZATION OF DIALYZED IRON-REACTIVE MUCOSUBSTANCE IN RABBIT HETEROPHILS, BASOPHILS, AND EOSINOPHILS

For ultrastructural localization of acid mucosubstances in rabbit granulocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde, or osmium tetroxide, sectioned at 40 µ, and stained with the Rinehart and Abul-Haj solution of dialyzed iron (DI). Heterophils revealed DI staining on the outer surface of the plasma membrane, in the Golgi complex involved in primary granulogenesis, and in primary granules. The intragranular distribution of DI-stained material varied at different stages in the maturation of primary granules. Immature granules of heterophils fixed by any of the three methods contained a peripheral concentric band of DI-positive material; however, fully mature primary granules possessed a core of DI-reactive material in heterophils fixed with osmium tetroxide, but they contained little or no staining in heterophils fixed with formalin or glutaraldehyde. Secondary granules of rabbit heterophils failed to stain with DI. Tertiary granules, observed only in late heterophils, contained distinct DI-positive particles. Basophil granules exhibited intensely DI-stained material distributed in an orderly pattern throughout the granule. In eosinophils, DI staining was localized in the Golgi complex and in the rims of a few immature cytoplasmic granules.