Nongraded Dehydration and Low-Pressure Infiltration for Rapid Celloidin Embedding of Brain Tissue

Various ways of shortening single steps in the celloidin process have been combined to form a routine method which may be completed, for tissues of average size, within a week following fixation. Fixed, washed tissue slices 5 mm thick are dehydrated in 1 or 2 changes of absolute ethanol and acetone, 1:1. This requires 24 hr in an incubator at 37 C, or 12-16 hr if a magnetic stirrer is used. After ether-alcohol for 4 hr. the tissues are transferred to 5% celloidin and infiltrated in a vacuum desiccator attached to a filter pump. When the volume of celloidin is reduced to half the original amount (about 2 hr), the tissues are removed from the infiltrating fluid and embedded in 10% celloidin. The blocks are hardened in chloroform and cleared by suspending them in 2 or 3 changes of terpineol agitated by a magnetic stirrer. Sections are cut in terpineol, using any type of microtome. After washing in 95% alcohol, they are mounted on albumenized slides for staining.     

A Rapid Plastic Embedding Technique for Preparation of Three-Micron Thick Sections of Decalcified Hard Tissue

A 24 hour start-to-finish method is described for the preparation of three-micronthick sections of decalcified hard tissues. Following acetone dehydration, the tissue to be embedded is infiltrated under vacuum with a series of graded clearing solutions which approach the content of the final methyl methacrylate mixture. After overnight in a 35 C oven, the plastic is polymerizd by four hours heating at 42 C. Three-micron-thick sections are then easily prepared using a Jung microtome for high resolution histologic or detailed autoradiographic procedures.     

Curdled Milk for Supporting Tissues in Celloidin Embedding

To make a material for supporting tissues during celloidin embedding, milk curd is hardened in ordinary strong alcohol, cut into suitable shapes, dehydrated, extracted with ether, and preserved indefinitely in 1:1 alcohol-ether. The material, which consists of the precipitated proteins of milk, is embedded and sectioned with the tissue. It presents no detectable opposition to the passage of the microtome-knife.     

Fluorescence of Plastic Embedded Tissue Sections After Curcumin Staining

The natural dye, curcumin (C.I. 75300) from turmeric, is obtained from the roots of Curcuma longa (Lillie 1977). Curcumin has scarcely been applied for histological work, and its fluorescence seems to have been overlooked. During the course of studies on fluorescent aluminum complexes (Del Castillo et al. 1987) we realized that this dye induces a green fluorescence of chromatin (Stockert et al. 1989). In this note we describe the fluorescence reaction of curcumin on semithin sections of olastic embedded tissues.     

Silver Protein Staining of Nerve Fibers in Celloidin Sections

Celloidin sections from formalin-fixed brain and spinal cord of primates are stored in 70% alcohol after cutting, soaked in 2% pyridine in 50% alcohol for 6-8 hr at 37 C, and transferred to 1% concentrated NH₄OH in 50% alcohol 15-18 hr at 20-25 C. After washing and flattening, the sections are transferred to 1% silver protein solution containing 30 ml of 0.2 M H₃BO₃/100 ml. Impregnation is accomplished in 50 ml screw-top jars, 50 mm in diameter, which are filled to a depth of 35 mm, and have 1 gm of copper foil, 0.002 inch thick added. The foil is folded in loose accordion-fashion, pierced and threaded, cleaned in 5% HNO₃, rinsed in distilled water, and suspended in the solution just above the sections by fastening the thread to the jar lid. The sections are impregnated for 24 hr at 37 C, rinsed in distilled water, reduced in a solution of 5% Na₂SO₃ and 1% hydroquinone for 10 min, washed in distilled water and toned in 0.2% gold chloride for 5 min. After rinsing in distilled water, the sections are transferred to 1% oxalic acid for 45-60 sec, washed in distilled water and placed in 5% Na₂S₂O₃ for 5 min. Sections are then washed, dehydrated to 95% alcohol, cleared in terpineol, followed by 3 changes in xylene, and mounted.     

Carbowax Embedding for Obtaining Thin Tissue Sections and Study of Intracellular Lipids

Carbowax, a water soluble wax, as an embedding agent is a valuable adjunct to the armamentarium of the tissue technologist. This report is intended to supplement previous publications on the use of Carbowax and to indicate die necessity for preheating and variation of Carbowax mixtures according to the climate.

Carbowax embedding provides an easy means for obtaining tissue sections 1 to 3 μ in thickness either with or without previous exposure to fat solvents. These sections are admirably suited for cytological study, particularly of intracellular lipoids.     

A Teflon Disk for Processing Loose Serial Celloidin Sections

Celloidin sections are routinely used for Nissl, Golgi, or Golgi-Cox staining (e.g., Glaser and Van der Loos 1981) when sections thicker than 30 μm are required. In spite of the advantages of the celloidin method (see Voogd and Feirabend 1981, Buschke 1979), processing free-floating serial sections of celloidin embedded material, which may often be preferred, is not very convenient.     

Rapid Dehydration for Celloidin Embedding by Means of Azeotropic Distiliation

By including plant material in a fractional distillation apparatus containing 95% alcohol and benzene it is possible to eliminate water completely in a constant boiling mixture. The dehydrated material can then be embedded in celloidin by orthodox techniques. In spite of the rapidity of the dehydration quite soft tissue as well as woody material can be processed. The method is cheap because it does not require absolute alcohol and is also suited to the humid climate in which it was developed.     

Gelatin Embedding of Central Nervous System Tissue Improves the Quality of Vibratome Sections

We have developed a procedure for Vibratome (Oxford Laboratories) sections that is particularly valuable for providing uniformly thick, well preserved CNS tissue sections for morphometric applications.     

Gelatin Embedding of Central Nervous System Tissue Improves the Quality of Vibratome Sections

We have developed a procedure for Vibratome (Oxford Laboratories) sections that is particularly valuable for providing uniformly thick, well preserved CNS tissue sections for morphometric applications.     

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.     

Light Microscopic Lectin Histochemistry on Celloidin Stabilized Cryostat Sections of Rat Colon

Celloidin stabilized pre-epithelial mucus gel (PMG) is stained in cryostat sections of the rat colon for light microscopic lectin histochemistry. Specific sugar residues of the mucins are demonstrated both in the PMG and in the mucin-containing cells of the mucosa.     

Histochemical Demonstration of Enzyme Activities in Plastic and Paraffin Embedded Tissue Sections

Histochemical staining for enzymes is usually performed on frozen sections. This report lists the longer incubation times required to demonstrate esterase, acid phosphatase, β-galactosidase, and cytochrome oxidase in plastic embedded and routine paraffin embedded tissues. The sections embedded in plastic, i.e. water soluble methacrylate (Polyscience's JB-4) and cut at 2 μm, were far superior to frozen Sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.     

Light Microscopic Lectin Histochemistry on Celloidin Stabilized Cryostat Sections of Rat Colon

Celloidin stabilized pre-epithelial mucus gel (PMG) is stained in cryostat sections of the rat colon for light microscopic lectin histochemistry. Specific sugar residues of the mucins are demonstrated both in the PMG and in the mucin-containing cells of the mucosa.     

A Protargol Method for Staining Nerve Fibers in Frozen or Celloidin Sections

Extensive experimentation with protargol staining of neurons in celloidin and frozen sections of organs has resulted in the following technic: Fix tissue in 10% aqueous formalin. Cut celloidin sections IS to 25 μ, frozen sections 25 to 40 μ. Place sections for 24 hours in 50% alcohol to which 1% by volume of NH₄OH has been added. Transfer the sections directly into a 1% aqueous solution of protargol, containing 0.2 to 0.3 g. of electrolytic copper foil which has been coated with a 0.5% solution of celloidin, and allow to stand for 6 to 8 hours at 37° C. Caution: In this and the succeeding step the sections must not be allowed to come in contact with the copper. From aqueous protargol, place the sections for 24 to 48 hours at 37° C. directly into a pyridinated solution of alcoholic protargol (1.0% aqueous solution protargol, 50 ml.; 95% alcohol, 50 ml.; pyridine, 0.5 to 2.0 ml.), containing 0.2 to 0.3 g. of coated copper. Rinse briefly in 50% alcohol and reduce 10 min. in an alkaline hydroquinone reducer (H₃BO₃, 1.4 g.; Na₂SO₃, anhydrous, 2.0 g.; hydroquinone, 0.3 g.; distilled water, 85 cc; acetone, 15 ml.). Wash thoroly in water and tone for 10 min. in 0.2% aqueous gold chloride, acidified with acetic acid. Wash in distilled water and reduce for 1 to 3 min. in 2% aqueous oxalic acid. Quickly rinse in distilled water and treat the sections 3 to 5 min. with 5% aqueous Na₂S₂O₃+5H₂O. Wash in water and stain overnight in Einarson's gallocyanin. Wash thoroly in water and place in 5% aqueous phosphotungstic acid for 30 min. From phosphotungstic acid transfer directly to a dilution (stock solution, 20 ml.; distilled water, 30 ml.) of the following stock staining solution: anilin blue, 0.01 g.; fast green FCF, 0.5 g.; orange G, 2.0 g.; distilled water, 92.0 ml.; glacial acetic acid, 8 ml.) and stain for 1 hour. Differentiate with 70% and 95% alcohol; pass the sections thru butyl alcohol and cedar oil; mount.