Serial Sections of Smears for Electron Microscopy

Ultrathin sections for electron microscopy may be prepared from smears or squashes embedded in methacrylate. The cover slip or glass slide with the attached fixed cellular material is passed through alcohols to methacrylate monomer and finally to monomer containing a catalyst. The portion of the smear to be sectioned is covered with a gelatin capsule containing partially polymerized methacrylate. When polymerization is completed at 47°C, the hardened block is separated from the cover slip and trimmed under the compound microscope so as to encompass the desired area. Photographs are made of the intact smear to afford a basis for identification of cellular materials in electron micrographs of the individual ultrathin sections.     

Histoautoradiographic Localization of Calcium in Oat Plant Tissues

The leaching of water-soluble and exchangeable calcium in histoautoradiog-raphy of oat tissue can be prevented by using acetone as the dehydration fluid (freeze substitution technique) and by keeping the tissue sections, while stretching on water, embedded in the methacrylate matrix. Ca⁴⁵ was either added to the mineral solution on which the oat plants were grown (75 μc), or applied on the leaf surface (8 μc). After freezing in melting isopentane, specimens of 1-2 mm dimensions are fixed for 24 hr in an acetone-OsO₄ (1%) solution at—80 C. Dehydration is obtained by transferring the material every day for 6 successive days to a fresh acetone solution at—80 C. The material is infiltrated by a three-time renewed monomer methacrylate mixture (methylmethacrylate I, butylmethacrylate 4) at—50 C. The specimens are embedded in the polymerizing methacrylate mixture at room temperature. Sections of 4-8 μ are easily cut with a rotating microtome. If the methacrylate is not removed from the sections, they can be stretched on water without leaching of calcium. The presence of methacrylate in no way hinders microscopic observation nor effective histoautoradiography.     

Notes of Technic

The leaching of water-soluble and exchangeable calcium in histoautoradiog-raphy of oat tissue can be prevented by using acetone as the dehydration fluid (freeze substitution technique) and by keeping the tissue sections, while stretching on water, embedded in the methacrylate matrix. Ca⁴⁵ was either added to the mineral solution on which the oat plants were grown (75 μc), or applied on the leaf surface (8 μc). After freezing in melting isopentane, specimens of 1-2 mm dimensions are fixed for 24 hr in an acetone-OsO₄ (1%) solution at—80 C. Dehydration is obtained by transferring the material every day for 6 successive days to a fresh acetone solution at—80 C. The material is infiltrated by a three-time renewed monomer methacrylate mixture (methylmethacrylate I, butylmethacrylate 4) at—50 C. The specimens are embedded in the polymerizing methacrylate mixture at room temperature. Sections of 4-8 μ are easily cut with a rotating microtome. If the methacrylate is not removed from the sections, they can be stretched on water without leaching of calcium. The presence of methacrylate in no way hinders microscopic observation nor effective histoautoradiography.     

A Plastic Embedding Medium for Thin Sectioning in Light Microscopy

Tissue blocks with surface areas up to 2 cm² can be sectioned at 1 or 2 μ after embedding in a medium consisting of: methyl methacrylate, 27 ml; polyethylene glycol distearate MW 1540, 6 gm; dibutyl phthalate, 4 ml; and Plexiglas molding powder A-100, 9 gm (added last). The methacrylate mixture is polymerized at 50° C by benzoyl peroxide, 0.8 gm/ 100 ml of methacrylate. The polymerized matrix is transparent and the blocks can be cut on a rotary microtome with a steel knife. The plastic can be removed from sections with acetone prior to staining. Artifacts caused by embedding and sectioning are negligible     

Double Emulsion Autoradiography for Identifying Tritium-Labelled cells in sections

The sensitivity of tissue autoradiography can be doubled and the number of false negative cells nearly eliminated by interposing thin tissue sections between two layers of photographic emulsion. A mouse was given 50 μc of tritiated thymidine (SA 2,500 c/M) intraperitoneally and killed 1.5 hr later. A portion of the small bowel was removed, fixed and embedded in methacrylate in the usual way. Sections 2 μ thick were cut and allowed to flatten on water at 40° C. Some sections were used to make conventional single emulsion auto-radiographs and other sections were interposed between two layers of emulsion by first coating slides with NTB 3 emulsion, picking up the sections from a water bath at 18° C, drying, soaking 1 min in benzene, drying, and then dipping again in NTB 3 emulsion. They were exposed at 4° C in a low humidity, 100% CO₂ atmosphere for 10 days, developed and covered in the usual way. There was an average of 20.16 ± 1.4 grains per labelled cell in the double emulsion group compared with 10.6 ± 0.9 grains in the single emulsion group. In the double emulsion autoradiographs there were 55.1 ± 1.65 labelled cells per unit area as compared with 39.8 2 2.0 in the single emulsion group.     

Enzyme immobilization by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperatures.

Enzyme immobilization by radiation-induced polymerization of hydrophilic glass-forming monomers, such as 2-hydroxyethyl methacrylate, was studied. Enzyme radiation damage could be sufficiently retarded at low temperatures. The immobilized enzyme activity yield was markedly higher at low temperature than at higher temperature polymerization. At low temperatures the polymerized composite had a porous structure owing to ice crystallization which depends on the monomer concentration. It was deduced that the enzyme was partially trapped on the polymer surface, partially isolated in the pore, and partially occluded inside the polymer matrix. A decrease in activity caused by enzyme leakage was observed with repeated use in enzyme reactions where the composites had a large porosity. The activity yield showed a maximum at certain optimum porosities, i.e., at optimum monomer concentrations. Continuous enzyme reaction was preferably carried out using immobilized enzyme columns.     

Staining Procedures for Ultrathin Sections of Tissues Embedded in Polyester Resin

Tissues were fixed for 30 min In cold (0-2° C) 1% OsO₄ (Palade) buffered at pH 7.7, to which 0.1% MgCl₂ was added. Dehydration was in a graded ethanol series (containing 0.5% MgCl₂) at 0-2° C, and terminated with 2 changes of absolute ethanol. Tissues were then transferred by a graded series to anhydrous acetone. Infiltration of the tissue with Vestopal-W (a polyester resin), is gradual with the aid of graded solutions of Vestopal-W in acetone. The infiltrated tissue is encapsulated and initial polymerization is done under ultraviolet light at room temperature for 8-16 hr. This is followed by final hardening at 60° C for 36-48 hr. Sections (0.2-1 μ) were cut, dried on slides, placed in acetone for 1 min and then treated by either of the following staining procedures: (1) Thionin-azure-fuchsin staining: Flood the preparation with 0.2% aqueous thionin and heat to 60-80° C for 3 min; if the preparation begins to dry, add stain. Rinse in distilled water. Flood the slide with 0.2% azure B in phosphate buffer at pH 9. Heat to 60-80° C for 3 min; do not permit the preparation to dry. Rinse in distilled water. Dip the slide in MacCallum's variant of Goodpasture's carbol-fuchsin stain for 1-2 sec. Rinse in distilled water. Check the preparation microscopically for intensity of the fuchsin stain. Repeat dips as may be needed to obtain the desired intensity. Rinse in distilled water. Dehydrate quickly in 95% and absolute alcohol; clear in 2 changes of xylene and cover in Permount or similar synthetic resin. (2) Thionin-azure counterstain for the periodic acid-Schiff reaction: Oxidize the tissue in 0.5% periodic acid for 15 min and transfer to Schiff's leucofuchsin solution for 30 min. Counterstain with 0.5% aqueous thionin for 3 min; wash in distilled water; stain in 0.2% azure B in phosphate buffer at pH 5.5; wash in distilled water; dehydrate; clear and cover as in the first method. For temporary preparations let dry after absolute alcohol and apply a drop of immersion oil directly on the section.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

N-butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18-24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

N-Butyl methacrylate and paraffin as an embedding medium for light microscopy.

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18--24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

Biotinylated glycopolymers synthesized by atom transfer radical polymerization

Biotinylated glycopolymers that bind to the protein streptavidin were synthesized by atom transfer radical polymerization (ATRP). Poly(methacrylate)s with pendent N-acetyl-d-glucosamines were prepared by polymerizing the protected monomer, followed by deprotection. Alternatively, the unprotected monomer was directly polymerized. Both paths provided well-defined glycopolymers with narrow molecular weight distributions (PDI = 1.07-1.23). The number-average molecular weights determined by gel permeation chromatography increased with increasing initial monomer-to-initiator ratios. The polymers were synthesized using a biotin-functionalized initiator for ATRP. Confirmation of the end group and binding to the protein streptavidin was achieved by (1)H NMR and surface plamon resonance.     

Embedding Media for 1-2 Micron Sectioning. 3. Hydroxyethyl Methacrylate-Benzoyl Peroxide Activated with Pyridine

A hydroxyethyl methacrylate (HEMA) monomer medium containing 2-butoxyethanol as the plasticizer requites only one stock solution consisting of: 50 ml of 94 or 96% HEMA containing 200 ppm inhibitor (Rohm and Haas, Philadelphia) is mixed with 12 ml 2-butoxyethanol; 0.25 gm benzoyl peroxide is added and permitted to dissolve at 20-25 C. This mixture is activated by the addition of 0.8-1.0 ml of pyridine. Polymerization of the activated mixture is initated in 15-20 hr at 25 C and in 2-4 hr at 50 C; polymerization of the mixture is complete in 2-3 days and 3-6 hr, respectively. The activated monomer mixture is stable at temperatures below 18 C; hence infiltration of tissues may be extended to 7-10 days by keeping the mixture at 0 to -40 C.     

Detection of S-Phase Cells in Smear Cytology Using in Vitro Bromodeoxyuridine Labeling

A technique Is described for rapid detection of S-pha?e cells of tumor tissues in smear specimens using bromodeoxyuridine (BrdU) immunostaining. Mouse NR-S1 tumors and human tumor specimens were prepared for smear cytology after incubation in RPMI 1640 culture medium containing 200 μM BrdU at 37 °C under 3 atm for 1 hr. Samples were fixed in 70% ethanol for 30 min and used immediately or air dried for 30 min. Samples were then denatured in either 4 N HC1 or 0.07 N NaOH to prepare partially single-stranded DMA. Fixation with air drying for 30 min followed by 30 min in 70% ethanol and 1 min denaturation with 0.07 N NaOH resulted in satisfactory staining quality. Cultured tumor specimens were processed for routine paraffin sections after smears were made for cytology. The labeling indices of the smear specimens and of the paraffin sections gave similar results. This technique should be useful in evaluating the cell proliferative potential of tumor tissue in smear cytology without processing paraffin sections.     

The Plastic Ethyl Methacrylate in Routine Laboratory Technic

The use of ethyl methacrylate as a permanent, transparent, colorless medium for mounting in toto invertebrate and vertebrate embryos and small adults is demonstrated.

The liquid monomer is partially polymerized by heating over a hot-plate, the reaction being aided by the use of benzoyl peroxide as a catalyst. The object to be mounted is fixed, stained, dehydrated, and cleared in the usual manner, and is then infiltrated with the partially polymerized ethyl methacrylate. It is oriented on a hardened base in a glass or porcelain dish, and the mold is then allowed to harden (polymerize) completely at a temperature of 38-40° C. under a 250-watt infra-red, ultra-violet bulb. After the block is removed from the dish it may be polished by the ordinary metallurgical methods. More than 40 species of animals have been satisfactorily treated in this manner.     

Determination of the maximum and minimum lethal temperatures (LT50) for Loxosceles intermedia Mello-Leitão, 1934 and L. laeta (Nicolet, 1849) (Araneae, Sicariidae)

In this study, the maximum and minimum lethal temperatures (LT₅₀) of L. intermedia and L. laeta were determined in two treatments: gradual heating (25–50°C) and cooling (25°C to −5°C), and 1 h at a constant temperature. In gradual temperatures change, L. intermedia mortality started at 40°C and the LT₅₀ was 42°C; for L. laeta, mortality began at 35°C and the LT₅₀ was 40°C. At low temperatures, mortality was registered only at −5°C for both species. In the constant temperature L. intermedia showed a maximum LT₅₀ at 35°C and L. laeta at 32°C; the minimum LT for both species was −7°C.     

Polyester Resin Embedding for Sectioning of Hard and Soft Tissues

Soft and calcareous tissues embedded in polyester resin may be cut on a sledge microtome to produce thin sections of 3-4 β thickness. Fixed tissues, dehydrated in ethyl alcohol, cleared in methyl benzoate and chloroform, are taken into a wide-necked bottle containing equal parts of polyester resin and chloroform with 0.75% catalyst. The bottle kept in water bath at 37°C is connected to a vacuum pump. With the evaporation of the chloroform under reduced pressure (approximately 10 mm Hg) infiltration is complete. Tissues transferred into a blocking form containing pure polyester resin with 1.5% catalyst are polymerized at 37° C until blocks are firm (48 hr or more). Blocks are prepared with at least 5 mm margin of plastic surrounding the tissue. The edge of the block adjacent to the knife is then filed at an angle of 45° to the cutting movement. Sections are cut with a wide-backed biplanar knife having a cutting edge of 40-44° positioned at an angle of 30° to the plastic block. As the resin is permeable to most stains, staining is carried out through the plastic Sections carried through staining procedures in wire baskets are floated onto slides and mounted in polystyrene; the cover-glass is compressed with a spring-clamp. Microscopic examination shows no staining of plastic, minimal shrinkage and good cellular detail.     

Embedding in Epoxy Resins for Ultrathin Sectioning in Electron Microscopy

Fixed tissue is dehydrated with tertiary butyl alcohol overnight. The following day it is cleared in toluene, infiltrated and embedded in Araldite resin-hardener-accelerator mixture without dibutyl phthalate, and polymerized at 60° C. More rapid than previous techniques, this method gives blocks which do not fracture unduly on trimming and provides sections of soft tissues at 1 μ for phase contrast microscopy, as well as ultrathin sections which cut as easily with glass knives as sections of methacrylate. Araldite manufactured in the U.S.A. and in England are different. Satisfactory proportions for the American are: hardener DDSA, 3.5 ml; casting resin 6005, 5.0 ml; accelerator B, 0.12 ml. For the British product, these are: hardener 964 B, 5.0 ml; casting resin M, 5.0 ml; accelerator 964 C, 0.25 ml. The use of 2% agar for orienting small specimens in Araldite is feasible. Mallory's borax-methylene blue has been applied to the staining of Araldite sections as thin as 0.5 μ mounted on glass slides.     

Differentiated Staining of Osmium Tetroxide-Fixed, Vestopal-w-Embedded Tissue in thin Sections

Tissues were fixed at 20° C for 1 hr in 1% OsO₄, buffered at pH 7.4 with veronal-acetate (Palade's fixative), soaked 5 min in the same buffer without OsO₄, then dehydrated in buffer-acetone mixtures of 30, 50, 75 and 90% acetone content, and finally in anhydrous acetone. Infiltration was accomplished through Vestopal-W-acetone mixtures of 1:3, 1:1, 3:1 to undiluted Vestopal. After polymerisation at 60° C for 24 hr, 1-2 μ sections were cut, dried on slides without adhesive, and stained by any of the following methods. (1) Mayer's acid hemalum: Flood the slides with the staining solution and allow to stand at 20°C for 2-3 hr while the water of the solution evaporates; wash in distilled water, 2 min; differentiate in 1% HCl; rinse 1-2 sec in 10% NH,OH. (2) Iron-trioxyhematein (of Hansen): Apply the staining solution as in method 1; wash 3-5 min in 5% acetic acid; restain for 1-12 hr by flooding with a mixture consisting of staining solution, 2 parts, and 1 part of a 1:1 mixture of 2% acetic acid and 2% H₂SO₄ (observe under microscope for staining intensity); wash 2 min in distilled water and 1 hr in tap water. (3) Iron-hematoxylin (Heidenhain): Mordant 6 hr in 2.5% iron-alum solution; wash 1 min in distilled water; stain in 1% or 0.5% ripened hematoxylin for 3-12 br; differentiate 8 min in 2.5%, and 15 min in 1% iron-alum solution; wash 1 hr in tap water. (4) Aceto-carmine (Schneider): Stain 12-24 hr; wash 0.5-1.0 min in distilled water. (5) Picrofuchsin: Stain 24-48 hr in 1% acid fuchsin dissolved in saturated aqueous picric acid; differentiate for only 1-2 sec in 96% ethanol. (6) Modified Giemsa: Mix 640 ml of a solution of 9.08 gm KH₂PO₄ in 1000 ml of distilled water and 360 ml of a solution of 11.88 gm Na₂HPO₄-2H₂O in 1000 ml of distilled water. Soak sections in this buffer, 12 hr. Dissolve 1.0 gm of azur I in 125 ml of boiling distilled water; add 0.5 gm of methylene blue; filter and add hot distilled water until a volume of 250 ml is reached (solution “AM”). Dissolve 1.5 gm of eosin, yellowish, in 250 ml of hot distilled water; filter (solution “E”). Mix 1.5 ml of “AM” in 100 ml of buffer with 3 ml of “E” in 100 ml of buffer. Stain 12-24 hr. Differentiate 3 sec in 25 ml methyl benzoate in 75 ml dioxane; 3 sec in 35 ml methyl benzoate in 65 ml acetone; 3 sec in 30 ml acetone in 70 ml methyl benzoate; and 3 sec in 5 ml acetone in 95 ml methyl benzoate. Dehydrated sections may be covered in a neutral synthetic resin (Caedax was used).     

Nile Blue Histochemical Method for Phospholipids

The technic recommended is: Fix 6-12 hr. in 10% formalin containing 1% CaCl₂. Cut frozen sections without embedding or after gelatin or carbowax. Stain 90 min. at 60°C. in saturated aqueous Nile blue sulfate, 500 ml. plus 50 ml. of 0.5% H₂SO₄, boiled 2 hr. before use. Rinse in distilled water, and place in acetone heated to 50°C. Remove the acetone from the source of heat and allow the sections to remain 30 min. Differentiate in 5% acetic acid 30 min., rinse in distilled water, and refine the differentiation in 0.5% HCl for 3 min. Wash in several changes of distilled water and mount in glycerol jelly. Results: phospholipids - blue; everything else - unstained. Counterstaining nuclei with safranin is optional, but if done, it preferably precedes the Nile blue and is then differentiated by the acetic acid. The histochemical principles on which the method is based are as follows: (1) The calcium compounds of phospholipids combine with the oxazine form of Nile blue sulfate and survive subsequent treatment; (2) neutral lipids are dissolved out by acetone; (3) proteins and other interfering substances are destained by the acetic acid and hydrochloric acid baths.     

Embedding Media for 1-2 Micron Sectioning. 2. Hydroxyethyl Methacrylate Combined with 2-Butoxyethanol

A hydroxyethyl methacrylate monomer medium incorporating 2-butoxyethanol requires 2 stock solutions for embedding. Solution A: 80 ml of hydroxyethyl methacrylate (Rohm and Haas Co., Philadelphia, Pa.) is mixed well with 16 ml of 2-butoxyethanol; 0.27 gm of benzoyl peroxide, the catalyst, is added and permitted to dissolve. Heating to 40-50 C may be used to accelerate its solution. Solution B: polyethylene glycol 200 or 400, 15 parts, and N,N-dimethylaniline, 1 part, are mixed thoroughly. Tissues are dehydrated in the customary manner to absolute ethanol or other comparable dehydrant, infiltrated completely with A, then cast in a mixture consisting of 42 parts of A well mixed with 1 part of B. Polymerizaion occurs in 4-7 hr. In a water bath at 20 C the time required was about 7 hr; at 28 C, 4 hr. This medium is based on the author's water-polyethylene glycol-hydroxyethyl methacrylate monomer medium (Stain Techn., 42: 119-23, 1967).