A Durable Nissl Stain for Frozen and Paraffin Sections

Frozen sections, 25-50 /j. thick, of formalin-fixed nervous tissues are mounted following the Albrecht gelatin technic. Paraffin sections, 15 p., are deparaffinized and transferred to absolute ethanol. The slides are then coated with celloidin. Both frozen and paraffin sections subsequently follow the same steps: absolute ethanol-chloroform (equal parts) for at least 20 min, 95% ethanol, 70% ethanol (1-3 min), then rinsed in distilled water. Sections are stained in Cresylechtviolett (Chroma) 0.5% aqueous solution containing 4 drops of glacial acetic acid per 100 ml, rinsed in distilled water, agitated in 70% ethanol until excess stain leaves the slide, and rinsed in 95% ethanol. Sections are then dehydrated in absolute ethanol, followed by butanol, cleared in xylene, and enclosed in permount.     

Application of the Papanicolaou Stain to Paraffin Sections

Routine paraffin sections from tissues fixed either in aqueous formalin, 80% alcohol (with or without 1% trichloracetic acid added), Carnoy's alcohol-chloroform-acetic (6:3:1) and Bouin's fixative were stained as follows: Harris' hematoxylin, 6 min; running water, 2-3 min; ascending grades of alcohol to 95%; orange G, 0.5% and phosphotungstic acid, 0.015% in 95% alcohol, 5 min; 95% alcohol, 2 changes; Papanicolaou's EA36, 2.5 min; dehydration, clearing, and covering in Permount. The results show morphology better than hematoxylin and eosin and the technic is recommended particularly for keratin, which always stains bright orange.     

A Methylene Blue-Basic Fuchsin Stain for Mast Cells in Paraffin Sections

In paraffin sections of rat tissue it is possible to stain mast cell granules blue in contrast to red nuclei, pale blue cytoplasmic ribonucleic acid, and colorless collagen. This is done by the following mixture: 1% methylene blue (pure, not polychrome), 9 ml; 0.1% basic fuchsin, 9 ml; glacial acetic acid, 2 ml. Stain formol-fixed, paraffin-processed sections for 5 min, wash in water and pass through acetone, 2 changes, 10 sec total, to xylene and a polystyrene mounting medium.     

The Papanicolaou Stain for Negri Bodies in Paraffin Sections

Paraffin sections ot the hippocampus (Amnion's horn) from brains of dogs and cows, fixed in sublimate-alcohol (HgCl₂, sat. aq., 1 vol.; absolute alcohol, 2 vol.) were stained by Papanicolaou's (1942) method for vaginal smears. Negri bodies were stained a bright rose color, with nucleoli dark blue. Even though the orange G were omitted, good staining of Negri bodies was obtained. Eliminating this dye simplifies the technic without impairing its effectiveness in the diagnosis of rabies, but the complete staining gives a somewhat more colorful and brighter histologic picture.     

Phosphotungstic Acid-Eosin Combined with Hematoxylin as A Stain for Negri Bodies in Paraffin Sections

Experimentation with the Papanicolaou stain in this laboratory led to the discovery that the eosin, combined with phosphotungstic acid, was responsible for differential staining of Negri bodies. Eosin prepared as in EA 36 was used, but without the light green and Bismarck brown. Paraffin sections of hippocampus from brains of animal affected with rabies were fixed in 10% formol or in a mixture of 2 volumes of saturated aqueous HgCl₂ and 1 volume of absolute alcohol. They were stained first with hematoxylin and then with eosin. This procedure gave better results than staining with other types of eosin or by the original EA 36 mixture. The Negri bodies were well stained and their structure easily visible. The best results were obtained from material fixed with the HgCl₂ solution.     

A Simplified Procedure for Preparation of Undecalcified Human Bone Sections

A new type of apparatus for sectioning samples of hard, undecalcified bone is described. Slices of fresh or archeological human bone 4-5 mm thick are dehydrated and then embedded in epoxy resin. The apparatus used to prepare sections from the resulting blocks consists of a low-speed rim-type diamond cut-off wheel and a slowly advancing table carrying the specimen held in a rotating mount. Sections may be cut at a thickness of 80 μm ± 1%. After cleaning in an ultrasonic bath, these can be mounted on slides for quantitative microscopic examination with transmitted light. Grinding and polishing are not necessary. The results obtained are illustrated.     

A Tetrachrome Stain for Fresh, Mineralized Bone Sections, Useful in the Diagnosis of Bone Diseases

Fresh, unprocessed bone is ground to sections 75-100 μ thick, stained in an aqueous solution composed of fast green FCF, 0.1 gm; orange G, 2.0 gm; distilled water, 100.0 ml; and adjusted to pH 6.65, then in a mixture of 1 part alcoholic solution of 0.25% celestine blue B and 9 parts of alcoholic solution of 0.1% basic fuchsin. Surface stain is removed by grinding sections to 50 μ and washing them in 1% invert soap (Zephiran) to remove adherent debris. (Commercial detergents and alkaline soaps may interfere with chromophore groups of the dyes.) Wash in tap water; rinse in distilled water and differentiate in 1% acetic alcohol. Dehydrate in ascending alcohols, clear in xylene and mount permanently in a neutral, synthetic resin. Active osteoid seams stain dark to light green; resting osteoid seams, red to bright orange red; transitional osteoid seams, geenish-yellow, orange red to red; older, partly mineralized matrix, orange; new, partly mineralized matrix, red; osteocyte nuclei, red; osteoblasts and osteoclasts, greenish-blue to dark purple nuclei and green or light green cytoplasm. Hyper-trophic and differentiating cartilage cells are stained light pink and dark red respectively. The staining reactions are consistent; the solutions are stable.     

A Thionin Stain for Visualizing Bone Cells, Mineralizing Fronts and Cement Lines in Undecalcified Bone Sections

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C.     

Serial Sections; Simplified Handling of Paraffin Ribbons on a Floating-Out Bath

Pieces of para & ribbon containing serial sections are arranged in overlapping rows on a microscope slide coated with albumen or glycerol. The assembly of sections is then floated free by immersing the slide in a bath of warm water. The rows of sections forming the assembled unit adhere to each other along their overlapping edges. After the sections have softened and expanded, the unit is picked up on a slide, covered with wet filter paper, rolled flat with a photographic print roller, and allowed to dry.     

Stain Technology a Bone Stain for Osteoid Seams in Fresh, Unembedded, Mineralized Bone

Fresh, ground, mineralized bone sections 75-100 μ thick are stained 90 minutes or 48 hours in the Bone Stain, a preparation containing fast green FCF, orange G, basic fuchsin, and azure II. Surface stain is then removed by grinding under running water. Sections are washed in 0.1% zephiran chloride (benzalkonium chloride) or in 0.01% mild soap and again washed in tap water, followed with distilled water. Sections are next differentiated in 0.01% acetic acid in 95% methanol, dehydrated in 95% ethanol and 100% ethanol, cleared in alcohol:xylene 1:1, 1:4, 1:9 and 2 changes of xylol, and then mounted permanently in Eukitt's mounting media.

Osteoid seams stain either green to jade green or red to dark red, incompletely mineralized bone red or orange yellow, and the zone of demarcation light green. The walls of lacunae, canaliculae, feathered bone, procedural artifacts and periosteocyte lacunar low-density versions stain red.

The method helps in the differential diagnosis of certain metabolic bone diseases in human biopsy and autopsy material.     

Modified Tetrachrome Method for Osteoid and Defectively Mineralized Bone in Paraffin Sections

A new modification of the tetrachrome method for bone osteoid in paraffin sections has been designed. The modified tetrachrome method suitable for routine use in any histology laboratory retains the simplicity of the original method and gives good results on the freshly fixed, decalcified, paraffin embedded material. Osteoid tissue is stained deep blue and normally mineralized bone is stained red. Defectively mineralized bone stains pale blue or pink and the cellular population is clearly identifiable. The ability to distinguish the osteoid tissue from mineralized bone and connective tissue and cartilage makes diagnosis of osteomalacia or osteoid producing tumors or assessment of ossification process straightforward, without the need for un-decalcified sections. By displaying simultaneously irregularities in the mineralized matrix and morphology of bone cells, the method also permits the diagnosis of conditions recently described in patients with osteoporotic fractures, such as osteocytic degeneration and bone tissue defects.     

A Method of Affixing Separate Thick Sections from Materials Embedded in Paraffin

Botanical studies often require thick histological sections (for embryology, pollen and spore arrangement in tetrads, etc.). Study of the original position of the generative cell in Angiosperms, for example (Huynh 1972), requires paraffin sections bearing entire pollen grains with a diameter of up to 80 μm. However, it is impossible to obtain ribbons with sections of such thickness. If the sections are affixed separately, they do not hold so strongly to slides as do those mounted as ribbons; this difficulty increases with thickness of section. in addition, affixing sections separately with the required order and spacing is tedious and difficult, demands a great deal of time, and even so, is not always successful. the simple method described here can remedy such inconveniences.     

Microwave-Stimulated Staining of Plastic Embedded Bone Marrow Sections with the Romanowsky-Giemsa Stain: Improved Staining Patterns

Staining plastic sections with the Romanowsky-Giemsa method is both time-consuming and difficult. This paper reports how the staining time can be reduced to 25 min using microwave irradiation of the staining solution. It is shown that staining results depend on the fixative used, staining temperature, dye concentration and pH of the staining solution as well as on several parameters of the microwave irradiation technique. The staining patterns are improved when compared with those obtained by conventional staining of plastic sections. The colors are more brilliant and greater contrasts are observed. Basophilia, polychromasia, and orthochromasia accompanying red cell maturation are more pronounced. For white cell maturation the initial appearance of specific granules (neutrophil, basophil, and eosinophil) is more evident. Thus, cell classification is easily accomplished using the described technique. It is suggested that microwave-stimulated staining be considered for routine use.     

A Simple Stain for Myelin in Frozen Sections: A Modification of Mahon's Method

A simple and rapid method for demonstrating myelinated nerve fibers in frozen sections of the central and peripheral nervous system is described. Material fixed by perfusion with mixed aldehydes gives the best results but the method also works on specimens fixed by immersion in formaldehyde. Frozen sections varying in thickness from 15-50 μm are mounted on slides subbed with chrome alum-gelatin. After hydration (60-140 min), Sections are mordanted (20-40 min) in 2.5% iron alum and rinsed briefly in three changes of distilled H₂O (total 2 min). Staining is for 60-180 min in 40 cc freshly made 10% alcoholic hematoxylin diluted with 165 cc distilled H₂O to which 15 cc saturated Li₂CO₂is added. the sections are washed in distilled H₂O (5-15 min) and dehydrated in graded alcohols without differentiation in mordant, and covered. Myelin stains a dark blue-purple against a light grey background. Fiber tracts, as well as individual myelinated fibers, are clearly demonstrated.     

A Method of Orienting Paraffin Sections for Three-Dimensional Reconstructions

To make reconstructions from serial sections, reference points are needed to orient the sections. Such points can be provided after paraffin embedding by cutting the bottom face of the block to form a plane and adding a groove along the center of this plane. The plane and groove are coated with Mimeograph Correction Fluid and the block is built up by dipping in hot paraffin so that the marked plane lies inside the block. Each section will have a blue line with a notch in it representing the plane and groove. This line remains through staining and is used to orient each section with respect to an eyepiece reticle. The reticle, in effect, supplies X and Y coordinates for every point in the specimen while the number of each section counted from one end is a Z coordinate.     

Stain Technology: A Combined Elastic, Fibrin and Collagen Stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified VerhoefPs elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by Hssamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsia, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

A Method for Processing Paraffin Sections of Multilayer Cultured Tissue

A simple and rapid method is described for processing histological preparations from multilayer cultures growing in plastic Petri dishes. A covering collodion film is utilized to remove the tissue from the plastic dish and transfer it onto a paper block prior to embedding in Paraplast. To avoid any disruption by the collodion of the plasticware, the cultured tissue is first immersed in a solution of collodion and absolute alcohol (1:1) and then covered with pure collodion. All steps are carried out in the cold. This procedure allows morphological, histochemical, immunofluorescent, and autoradiographic studies to be carried out on serial sections of cultured tissue.     

Penetration of Stain in Ultrathin Sections of Tobacco Mosaic Virus

Purified preparations of tobacco mosaic virus (TMV) and pieces of tomato leaves infected with TMV were embedded in methacrylate or epoxy resin, sectioned, and stained with 1.0% strontium permanganate for electron microscopy. In sections containing purified and intracellular virus, the apparent length of stained particles varied directly with section thickness, indicating stain penetration beyond the surface of the section. Penetration was demonstrated also by stereoscopy. Penetration was less complete when sections were allowed to dry before staining. In most instances the number of identifiable particles per unit area was independent of section thickness but increased when both surfaces of the sections were stained instead of only one surface. Staining was prevented by thin films of methacrylate or epoxy resin placed between the virus section and staining solution. Most results supported the view that electron scattering capacity was enhanced only in particles which intersected the surface of the section exposed to permanganate.     

Two Methods for Flat Embedding Sections in LR White Cut from Paraffin Blocks

Two simple methods are described for flat embedding of sections cut from paraffin blocks of brain tissue in the hydrophilic resin LR White. The ability of fresh resin to polymerize when added to already cured resin is exploited. In the first method, a tissue section with a drop of LR White is pressed to the bottom of a gelatin capsule using a blank block. In the second method, the section in a drop of resin is sandwiched between the sawed halves of a blank block. After curing, thin sections containing large areas of tissue can be collected and processed for immunocytochemistr y.