Embedding with Low Viscosity Nitrocellulose

The gelation and cutting of embedding masses of low viscosity nitrocellulose (L. V. N.) and of celloidin were compared. L. V. N. forms a firmer mass which can be cut into thinner sections than celloidin. It tolerates considerable water (up to 6%) in a solvent system of alcohol-water-ether, thereby permitting the use of 95% alcohol instead of absolute for making up the solution. The fluidity of its solutions permits transfer of tissue directly from alcohol-ether to a 20% embedding solution. Faults of L. V. N. are: the nitrated cotton lint contains some grit (hence its solution should be allowed to settle), the sections cut from it are somewhat more easily torn than celloidin sections, and it is sufficiently soluble in absolute alcohol to preclude the use of this fluid in handling sections.     

Embedding with Low Viscosity Nitrocellulose

The gelation and cutting of embedding masses of low viscosity nitrocellulose (L. V. N.) and of celloidin were compared. L. V. N. forms a firmer mass which can be cut into thinner sections than celloidin. It tolerates considerable water (up to 6%) in a solvent system of alcohol-water-ether, thereby permitting the use of 95% alcohol instead of absolute for making up the solution. The fluidity of its solutions permits transfer of tissue directly from alcohol-ether to a 20% embedding solution. Faults of L. V. N. are: the nitrated cotton lint contains some grit (hence its solution should be allowed to settle), the sections cut from it are somewhat more easily torn than celloidin sections, and it is sufficiently soluble in absolute alcohol to preclude the use of this fluid in handling sections.     

The Rapid Preparation of Celloidin Serial Sections Following India Ink Injections

For the study of capillary penetration in the central nervous system of the chick embryo, following India ink injections, celloidin serial sections are superior to those prepared by the paraffin technic. The celloidin sections are arranged on a moist cigarette paper mat, which when filled is inverted and applied to a microscope slide so that the sections contact the glass surface. Subsequent to dehydration and clearing the sections are isolated on the slide by peeling off the cigarette paper. Forty-five minutes are required to prepare a slide of thirty sections from the time the block is trimmed until the cover slip is mounted with Clarite.     

A Simplified and Convenient Method for the Double-Embedding of Tissues

A method for embedding tissues with a celloidin-paraffin combination is presented. The essential features of the process depend upon (1) a thorough infiltration of the specimen with celloidin of low concentration, and (2) the subsequent impregnation of both the specimen and the celloidin with paraffin.

The methods for sectioning, and the removal of the embedding agent are given.

The chief advantages of this method are: the preservation of all of the advantages of celloidin embedding but with a great saving of time, and greater convenience of storage; the cutting of thin sections (2μ for many types of tissues); it is useful for embedding specimens for which neither pure paraffin nor pure celloidin are entirely satisfactory, i.e. those containing tissues differing in density.     

Plastic Embedding of thick Celloidin Sections of nerve Tissue

A method is described for mounting Golgi-impregnated and Weigert-stained thick celloidin sections of brain and spinal cord in transparent plastic. Finished mounts have good optical properties and are suitable for macroscopic and microscopic observation. The durability of such preparations makes them superior to similar material prepared by the more conventional methods. Holes of suitable size were cut in matrices of 2.5 × 5 × 3/16 inches Plexiglas. Ward's Bio-plastic was used to form a base for the holes and also as the embedding medium for the sections. Plate glass formed a working substrate and gave a polished surface to the plastic base and later to the top of the preparation. For Golgi material (200μ) the celloidin was removed by dioxane. A dioxane-plastic bath preceded plastic embedding. For Weigert material (30-40μ) celloidin was not removed due to fragility of sections. Prior to plastic embedding, they were subjected first to benzol and then to a benzol-plastic bath.     

Sectioning Tough Stems Infiltrated with Polyethylene Glycol

Herbaceous stems eventually become too tough for paraffin sections. Alternative celloidin and plastic sections are laborious and expensive to make. Sticky or corrosive sap precludes fresh sections. These restrictions have been circumvented with a new method.     

Demonstration of cathepsin B in the rat kidney using fluorescence histochemistry

The procedure of mounting freeze-dried sections with celloidin was adapted for the fluorescent-histochemical demonstration of cathepsin B in the rat kidney. A good localization of reaction products was shown in freeze-dried, 5-micron sections which had been mounted free floating with 1.5% celloidin solution on albuminized slides. Using this procedure, the reaction products were localized in the lysosomes, particularly those of the convoluted proximal tubule.     

Celloidin as a protective film for improving the histochemical localization of succinate dehydrogenase

Synopsis The effect on the localization of succinate dehydrogenase of coating fresh and frozen-dried cryostat sections of unfixed hamster liver with celloidin was examined. It was found that the protective celloidin film not only leads to a more discrete localization of the enzyme, but is also prevents high losses of nitrogenous materials from sections into the incubation medium, a 30% loss in contrast to the 70% with fresh, non-coated sections. Further, after incubation, the morphology of the coated sections is much better than the uncoated ones.     

A new technique for staining sections and recovering celloidin film in high resolution autoradiography

Ultrathin sections are stained immediately after cutting by placing them in contact with staining solution and then placed on a slide covered by a celloidin film. This method largely avoids precipitates of heavy metals. The recovering of celloidin film is improved using a stainless steel basket. This technique is far more reliable than that involving use of a filter paper.     

Rapid method of silver nitrate impregnation of elements of the peripheral nervous system suitable for celloidin and paraffin sections

According to the method of neural elements impregnation in the authors' modification, the object is fixed for 6-12 h in Lillie fluid cooled to 4 degrees C. Then the object is kept under tap water for 2-6 h. Frozen sections are prepared and kept in pure pyridine for 1-6 h. When the sections are embedded into paraffin or celloidin, they are put into alcohol solutions gradually decreasing their concentration until water is reached, then put into pyridine. In order to remove cellulose, the celloidin sections are treated in 3 portions of pyridine (in the 1st and 2nd-for 10 min, and in the 3d-for 6 h). Then they are washed under tap water for 2-4 h and in distilled water for 30-40 min. Further treatment is performed according to the methods by Bielschowsky - Gros, Kampos or Rasskazova. Excess silver is removed by treating the sections in 2% ammonium persulfate under the microscope control (the process is stopped by putting the sections into 7% sodium hyposulfate for 10 min). Then the sections are treated in 0.1% aurum chloride, in 5% hyposulfite to reveale the tissue background [corrected] and by means of routine histological techniques either after Brashet, Hale, PAS-positive reaction or other methods applied after fixation in Lillie fluid.     

A Rapid Celloidin Method for the Rotary Microtome

A method is described which combines the writer's hot celloidin technic¹ with a form of the clearing-before-cutting procedure. The method requires only 16-17 days and yields a block which may be cut in any microtome, the sections being as thin as those afforded by paraffin with comparable material. The advantages of celloidin over paraffin, listed in the writer's earlier paper, are retained in the present method which, altho consuming more time than the hot process, requires less skill and gives superior results.     

An Improved Fixing Solution for Methylene Blue Preparations

A method is described which combines the writer's hot celloidin technic¹ with a form of the clearing-before-cutting procedure. The method requires only 16–17 days and yields a block which may be cut in any microtome, the sections being as thin as those afforded by paraffin with comparable material. The advantages of celloidin over paraffin, listed in the writer's earlier paper, are retained in the present method which, altho consuming more time than the hot process, requires less skill and gives superior results.     

Notes on Technic: A Device for Centering Tissue Blocks on the Porter-Blum Microtome

A method of double embedding fixed tissues in 3% low viscosity nitrocellulose and paraffin is described. Five percent phenol in 80% alcohol during dehydration and 5% glycerin in the nitrocellulose solutions enhance cutting qualities. A modified Ruyter's solution is used to flatten sections. After a section is aflixed to a slide, it is passed through chloroform and acetone to remove the paraftin and celloidin. A 1% celloidin dip insures adherence of the seaion to the slide. Slides are stored in 70% alcohol until they are to be stained. Following staining and dehydration in graded alcohols, clearing should be done in a 1: 3 mixture of terpineol and toluene.     

Double Embedding Again

A method of double embedding fixed tissues in 3% low viscosity nitrocellulose and paraffin is described. Five percent phenol in 80% alcohol during dehydration and 5% glycerin in the nitrocellulose solutions enhance cutting qualities. A modified Ruyter's solution is used to flatten sections. After a section is aflixed to a slide, it is passed through chloroform and acetone to remove the paraftin and celloidin. A 1% celloidin dip insures adherence of the seaion to the slide. Slides are stored in 70% alcohol until they are to be stained. Following staining and dehydration in graded alcohols, clearing should be done in a 1: 3 mixture of terpineol and toluene.     

Wrinkle-Free Plastic Sections for Light Microscopy

Plastic sections 0.5 to 2 μm thick are routinely used for light microscopy. Although plastic sections have several advantages over paraffin or celloidin sections, a problem that is often encountered with plastic sections is wrinkling (Fig. 1). Wrinkling occurs during staining when sections dried on glass slides are covered with stain and heated to hasten the penetration of the stain. Mounted sections heated on glass slides, but not stained, ordinarily lack wrinkles, even when examined with phase contrast optics. Similarly, mounted sections covered with stain, but not heated, lack wrinkles; unfortunately, such sections fail to stain adequately. Unmounted sections floated on heated drops of stain also lack wrinkles (Millonig 1980). Thus, it is clear that wrinkling occurs only when mounted sections are covered with stain and heated.     

Quick Staining Method For Frozen Sections

Since the advent and general acceptance of frozen sections in histological and pathological laboratories it has been necessary to devise methods for staining these sections. The usual method is fixing the tissue to a slide by the use of celloidin. This paper is an attempt to describe a permanent, quick method of staining frozen sections without distortion or mechanical tearing of the tissues.     

A method for obtaining histological sections from tissue previously studied by scanning electron microscopy

An accelerated method of paraffin embedding of tissue specimens previously examined with scanning electron microscopy is proposed aimed to obtain sections for routine histological examination. The tissue is passed through acetone, absolute alcohol, alcoholic-oil celloidin solution, chloroform to be eventually mounted into paraffin. The method allows obtaining good quality sections within 24 hours.     

Deplasticizing of thick Epon sections involving a new adhesive technique and staining of nervous tissue

The present communication deals with a technique developed for the selective staining of neural tissue in thick (10 micron) Epon sections. A new adhesive method was needed, because the known techniques are only applicable to 0.5-2 micron thin sections. The critical step in the procedure is the adhesion of the sections onto the slides. This is accomplished by heating the sections on top of a uniform layer of albumin glycerol on the slide followed by coating with celloidin. The results after deplasticizing and coagulation with this technique are comparable to those obtained by paraffin or frozen section techniques, but in addition have the advantage of Epoxy resin embedding e.g. the possibility of cutting undecalcified hard tissues and sections for serial reconstruction.     

Mounting Frozen Sections with Gelatin

Frozen sections, 15-50 µ thick, are soaked for 5 minutes or longer in a mixture of equal parts of 1.5% aqueous gelatin and 80% alcohol, and teased onto a slide. After allowing excess fluid to evaporate, sections will be moist and can be blotted with filter paper that may require dampening with 95% alcohol. Immersed in 95% alcohol, the remaining gelatin will congeal, anchoring the section to the slide. If necessary, the sections can subsequently be coated with celloidin.     

Factors which Influence the Latent Image in Autoradiography

Sections treated with N-ethyl-maleim'ide, a sulfhydryl blocking reagent, exhibit similar grain counts over nonradioactive and S-labeled tissues. This reveals that no chemo-graphic effects were produced by sulfhydryl groups in tissues fixed and prepared in the manner described. It was noted, however, that background count over tissue sections is consistently tower than over adjacent him, indicating perhaps the existence of substances in tissues which interfere with the production of the latent image. These effects have been eliminated by coating the tissues with celloidin. Celloidin coating can be used for most emitters including S but not for tritium. A special method for tritium, not involving celloidin coating is given.