Staining method for whole-body autoradiography.

Sagittal whole-body sections of frozen mice were cut on a hydraulicly driven microtome in a cryostat at--15 C by applying cotton or nylon-backed adhesive tape to the mouse before cutting. Section thickness was 20 mu. The sections, still adhering to the tape, were dried in the cryostat (-15C) under atmospheric pressure. After autoradiography, the sections were pressed to a glass slide spread with a mixture of albumin and glycerin. The slide was immersed in xylene at 30 C for 15 min. The tape was then removed from the slide, where the section remained to be stained with hematoxylin-eosin. The section thus obtained enabled the tissue histology to be related to the autoradiogram. This method may also be applied to histochemical studies of substances insoluble in xylene.     

Thin Sections : I. A Study of Section Thickness and Physical Distortion Produced during Microtomy

Knowledge of the thickness of sections is important for proper interpretation of electron micrographs. Therefore, the thicknesses of sections of n-butyl methacrylate polymer were determined by ellipsometry, and correlated with the color shown in reflected light. The results are: gray, thinner than 60 mµ; silver, 60 to 90 mµ; gold, 90 to 150 mµ; purple, 150 to 190 mµ; blue, 190 to 240 mµ; green, 240 to 280 mµ; and yellow, 280 to 320 mµ. These results agree well with optical theory and with previous published data for thin films. Sections, after cutting, are 30 to 40 per cent shorter than the face of the block from which they were cut. Only a small improvement results from allowing the sections to remain in the collecting trough at room temperature. Heating above room temperature, however, reduces this shortening, with a corresponding improvement in dimensions and spatial relationships in the sections. When the thickness of the section is considered in interpreting electron micrographs instead of considering the section to be two-dimensional, a more accurate interpretation is possible. The consideration of electron micrographs as arising from projections of many profiles from throughout the whole thickness of the section explains the apparent lack of continuity often observed in serial sections. It is believed that serial sections are actually continuous, but that the change in size of structure through the thickness of one section and the consideration of only the largest profile shown in the micrograph can account for the lack of continuity previously observed.     

Thin sections. I. A study of section thickness and physical distortion produced during microtomy

Knowledge of the thickness of sections is important for proper interpretation of electron micrographs. Therefore, the thicknesses of sections of n-butyl methacrylate polymer were determined by ellipsometry, and correlated with the color shown in reflected light. The results are: gray, thinner than 60 mmicro; silver, 60 to 90 mmicro; gold, 90 to 150 mmicro; purple, 150 to 190 mmicro; blue, 190 to 240 mmicro; green, 240 to 280 mmicro; and yellow, 280 to 320 mmicro. These results agree well with optical theory and with previous published data for thin films. Sections, after cutting, are 30 to 40 per cent shorter than the face of the block from which they were cut. Only a small improvement results from allowing the sections to remain in the collecting trough at room temperature. Heating above room temperature, however, reduces this shortening, with a corresponding improvement in dimensions and spatial relationships in the sections. When the thickness of the section is considered in interpreting electron micrographs instead of considering the section to be two-dimensional, a more accurate interpretation is possible. The consideration of electron micrographs as arising from projections of many profiles from throughout the whole thickness of the section explains the apparent lack of continuity often observed in serial sections. It is believed that serial sections are actually continuous, but that the change in size of structure through the thickness of one section and the consideration of only the largest profile shown in the micrograph can account for the lack of continuity previously observed.     

Alcoholic Bouin fixation of insect nervous systems for bodian silver staining. III. A shortened, single impregnation method

Satisfactory Bodian silver staining of paraffin wax sections of both locust (Schistocerca gregaria) and cockroach (Periplaneta americana) central nerve tissue can be obtained with only one impregnation, instead of the usual two, by the following modified procedure. Freshly dissected ganglia are fixed in an improved synthetic alcoholic Bouin (40% formaldehyde 0-15: ethanol 25: acetic acid 5: picric acid 0.5: either ethyl acetate 5 and diethoxymethane 15, or ethyl acetate 25: distilled water to 100). Formaldehyde content governs intensity of glial staining (little or none without formaldehyde) and the mixture with more ethyl acetate substituted for diethoxymethane gives more intense staining overall. Sections are impregnated once only, overnight, in 2% Protargol solution brought to about pH 8.4 with ammonium hydroxide and containing 1.3 g of copper per 65 ml. Depending on fixative composition, species, section thickness and contrast desired between nerve fibers and background, the subsequent distilled water rinse is shortened or omitted and sections are developed in 1% hydroquinone with sodium sulfite content reduced (to 2.5-4% Na2SO3.7H2O) for thinner (10 micrometer) sections but normal (10%) for thicker (20 micrometer) ones. Sections are finally washed, gold intensified, treated with sodium thiosulfate and dehydrated, cleared and mounted as usual. Results are slightly lighter than with normal double impregnation but entirely suitable for studies of neuroanatomy.     

Producing Frozen Sections of Calcified Bone

We describe a procedure for the rapid production and maintenance of fresh frozen bone biopsies which can be used for a variety of immunohistochemical techniques. Within 5 min of excision. tissue is placed in cold 5% polyvinyl alcohol, surrounded with 3% carboxymethylcel-lulose in a hand made aluminum foil embedding mold and frozen by immersion in an absolute ethanol/dry ice slurry at -70 C. The tissue block is attached to the specimen stub with cryocom-pound and installed in a -32 C cryostat whose tungsten carbide D profile knife is maintained at -70 C. Automatic controls are set at a slow cutting speed and the “sectioning window” is adjusted to fit the biopsy size. Knife angle, thickness gauge and antiroll bar are changed to produce a complete section. The block face is smoothly “papered” with a polyvinylpyrrolidone (PVP) impregnated Ross lens paper strip. A single section is cut and positioned on a sequentially numbered, acid cleaned, double dipped chrome-alum gelatin coated slide: adhesion is aided by “press-blotting” with bibulous paper. Sections are stored at -20 C or in a desiccator at room temperature. A brief fixation followed by removal of the water soluble PVP and lens paper generates fresh frozen bone sections suitable for further analysis.     

Alcoholic Bouin Fixation of Insect Nervous Systems for Bodian Silver Staining. III. A Shortened, Single Impregnation Method

Satisfactory Bodian silver staining of paraffin wax sections of both locust (Schistocerca gregaria) and cockroach (Periplaneta americana) central nerve tissue can be obtained with only one impregnation, instead of the usual two, by the following modified procedure. Freshly dissected ganglia are fixed in an improved synthetic alcoholic Bouin (40% formaldehyde 0-15:ethanol 25:acetic acid 5: picric acid 0.5:either ethyl acetate 5 and diethoxymethane 15, or ethyl acetate 25:distilled water to 100). Formaldehyde content governs intensity of glial staining (little or none without formaldehyde) and the mixture with more ethyl acetate substituted for diethoxymethane gives more intense staining overall. Sections are impregnated once only, overnight, in 2% Protargol solution brought to about pH 8.4 with ammonium hydroxide and containing 1.3 g of copper per 65 ml. Depending on fixative composition, species, section thickness and contrast desired between nerve fibers and background, the subsequent distilled water rinse is shortened or omitted and sections are developed in 1% hydroquinone with sodium sulfite content reduced (to 2.5-4% Na₂SO₃·7H₂O) for thinner (10 μm) sections but normal (10%) for thicker (20 μm) ones. Sections are finally washed, gold intensified, treated with sodium thiosulfate and dehydrated, cleared and mounted as usual. Results are slightly lighter than with normal double impregnation but entirely suitable for studies of neuroanatomy.     

A method for the preparation of serial thick sections of plastic-embedded plant tissues.

A procedure is described in which thick sections (2-10 mu or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, alkalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pre-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Improvements in histological techniques for epoxy-resin embedded bone specimens

A procedure is presented in which some of the processing difficulties with fixation, embedding and cutting whole mouse bones and large bone pieces from other species are considered. The bone specimens are fixed in acetone or by a Karnovsky-formol-saline process which preserves intact endosteal surface-to-cortex layers. After fixation the bones are embedded in a hard mixture of epoxy resin to provide blocks with face sizes up to 3.5 x 3.0 cm. Mineralized sections are cut to 4 micrometer; demineralized at 3 micrometer. Sections are fastened to gelatin-subbed slides with pressure plates which produce flat, secure sections. After removal of the plastic, an unmodified Mayer's hematoxylin and a polychromatic eosin staining method is applied to demineralized sections, and a slightly modified method to mineralized sections.     

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.     

Polyethylene Glycol Distearate 600 With 10% 1-Hexadecanol; A Superior Embedding Wax for WARM Climates

A mixture of 90% polyethylene glycol distearate 600 and 10% 1-hexadecanol has a melting point of approximately 43 C and is suitable as a replacement for conventional polyester wax where laboratory temperatures are above 24 C. Sections are attached to slides by floating out on 10% formalin and picked up on slides precoated with undiluted egg albumen. Since the wax is opaque, very small specimens can be embedded after fixation in a block of 0.5% agar before dehydration and embedding in wax, thus facilitating their handling and orientation. The wax mixture sections well with a razor blade in a holder.     

Sections from double mesas obtained at the same tissue plane

A method for obtaining sections from two areas in the face plane of a tissue block is described. It facilitates ultrathin sectioning where virtually identical planes of section are essential but where areas of interest are too far apart to be included in a single section. Two horizontally separated mesas are prepared; sections are cut from the first with the knife rotated around its vertical axis by 2-3 degrees to provide clearance for the other. The second mesa is then sectioned with the knife rotated 4-6 degrees in the opposite direction. Similarly, by changing the vertical inclination of the block, two additional vertically separated mesas can be cut. This procedure is of great value for comparative morphometric studies of material from opposite sides of individual specimens.     

Sections from Double Mesas Obtained at the Same Tissue Plane

A method for obtaining sections from two areas in the face plane of a tissue block is described. It facilitates ultrathin sectioning where virtually identical planes of section are essential but where areas of interest are too far apart to be included in a single section. Two horizontally separated mesas are prepared; sections are cut from the first with the knife rotated around its vertical axis by 2-3° to provide clearance for the other. The second mesa is then sectioned with the knife rotated 4-6° in the opposite direction. Similarly, by changing the vertical inclination of the block, two additional vertically separated mesas can be cut. This procedure is of great value for comparative morphometric studies of material from opposite sides of individual specimens.     

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.     

Improvements in Histological Techniques for Epoxy-Resin Embedded Bone Specimens

A procedure is presented in which some of the processing difficulties with fixation, embedding and cutting whole mouse bones and large bone pieces from other species are considered. The bone specimens are fixed in acetone or by a Karnovsky-formol-saline process which preserves intact endosteal surface-to-cortex layers. After fixation the bones are embedded in a hard mixture of epoxy resin to provide blocks with face sizes up to 3.5 × 3.0 cm. Mineralized sections are cut at 4 μm; demineralized at 3 μm. Sections are fastened to gelatin-subbed slides with pressure plates which produce flat, secure sections. After removal of the plastic, an unmodified Mayer's hematoxylin and a polychromatic eosin staining method is applied to demineralized sections, and a slightly modified method to mineralized sections.     

Differentiation of Myofibrillae, Reticular and Collagenous Fibrils in Vertebrates

A procedure for the differentiation of the mesenchymal derivatives, myofibrillae, reticular and collagenous fibers is presented. Formol-Zenker fixation (5-12 hours) is followed by the washing, iodinization, dehydration and paraffin embedding steps routine for that fixative with the following modifications. Zirkle's butyl alcohol series is used for dehydration and infiltration with paraffin as well as in the alcohol slide series. Embedding paraffin used is Parawax plus 8-10% bayberry wax. Tissue-exposed surface of paraffin block is soaked in water overnight before cutting serial sections at 3-5μ. Sections are mounted using the dilute albumen method, and the slides, thoroughly dried at 37^oC. overnight, are left at 60^o for 10 minutes to melt the paraffin of the sections. Before staining, the sections are given a preliminary treatment with potassium permanganate and oxalic acid. For reticular staining a 10% silver nitrate bath is succeeded by an ammoniacal silver carbonate solution followed by reduction in 1% neutral formalin, toning in gold chloride and fixing in sodium thiosulphate. Myofibrillae, the sacroplasmic limiting membrane and other sarcous elements are stained by Heidenhain's azocarmine solution, adult tissues at room temperature and fetal tissues at 50 ^oC. Differentiation in phosphotungstic acid is followed by the staining of collagenous fibers. For adult tissue, light green SF (C.C.) is used and for fetal tissue, fast green FCF (C.C). A discussion of the preparation of ammoniacal silver solutions is included. Both stock and used solutions of ammoniacal silver have been in use by the author for over a period of two years.     

A Method for the Preparation of Undecalcified Bone Sections for Light Microscopy and Microradiography

A method which gives good quality 1-2 μm thick sections of undecaldfied cancellous and thin cortical bones for light miuoscopy is described. Formalin fixed material is dehydrated in graded acetones and embedded in a modiEed formula of Spurr's low viscosity embedding medium. After a 16 hour polymerisation period at 60 C, sections are cut at 1-2 μm thickness on a Porter-Blum JB4A rotary microtome Using glass knives. Sections are attached to clean glass slides with heat, the resin degraded in bromine vapour and removed in acetone. This allows comparative ease of staining. The technique is rapid, does not interfere with tetracycline fluorescence and the same specimens can be used to prepare thick sections for microradiography.     

Differential Staining Of Tissue In The Block With Picric Acid And The Feulgen Reaction

The authors have found a modification of the Feulgen reaction to be a satisfactory stain for tissue in the block.

Pieces of fresh mammalian tissue not thicker than 5 mm. are fixed for approximately 48 hours at 25° C. in a mixture of equal parts of 5% aqueous sulfosalicylic acid and saturated aqueous picric acid. They are washed for 30 minutes in three ten-minute changes of distilled water and placed in Feulgen's staining solution diluted to one-half strength with distilled water. The staining solution is allowed to act for 24 hours (2 to 3 mm. thick blocks) up to 48 hours for 5 mm. thickness. After staining, the specimens are transferred to a mixture of sodium bisulfite, 0.5 g. and N hydrochloric acid, 5 ml. in' 100 ml. of distilled water. Two changes of IS to 30 min. each in the acid sulfite are given and these are followed by dehydration through 50%, 70% and 95% alcohol. One to two hours are allowed for each change except the last 95%, in which the stained tissue is allowed to remain overnight. The dehydration is completed in two changes of absolute alcohol with subsequent clearing in xylene and embedding in paraffin. Sections may be cut 10 μ or other thickness desired, mounted on slides, paraffin removed, and covered in the usual manner. Nuclei stain reddish violet against a lemon yellow background when the stain is typical. Orange G, 200 mg. per 100 ml. may be added to the fixing fluid if a more polychromatic effect is desired.     

A method for preparing whole-body sections suitable for autoradiographic, histological and histochemical studies

A method for the preparation of whole-body sections suitable for autoradiographic and histochemical study is described. Radioactive calcium chloride or [14C]proline was injected into the abdominal cavity of a rat. Thirty-five minutes after injection of calcium chloride or 40 min after injection of proline the rat was frozen in a mixture of hexane and solid carbon dioxide and blocked in 5% sodium carboxymethyl cellulose. The carboxymethyl cellulose block was trimmed and a piece of copy paper was attached to the surface of the block with cellulose tape. Cryotome sections cut from the block were transferred from the paper to a glass slide coated with synthetic rubber adhesive. For whole-body autoradiography, sections were freeze-dried for 2 days and then placed against X-ray film. For light microscopic autoradiography, the freeze-dried sections were covered with a dried film of photographic emulsion. For histochemical use, the sections were fixed by raising the temperature to 4 C after immersion in 100% ethanol below -10 C. For histological observation, sections were postfixed with 2.5% glutaraldehyde and stained. Whole-body and light microscopic autoradiographs showed that sections so prepared could be used for the demonstration of soluble substances in whole-body sections and for detailed autoradiography at the light microscopic level, and the stained sections could be used for histological and histochemical studies.     

A Method for Preparing Whole-Body Sections Suitable for Autoradiographic, Histological and Histochemical Studies

A method for the preparation of whole-body sections suitable for autoradiographic and histochemical study is described. Radioactive calcium chloride or [¹⁴Clproline was injected into the abdominal cavity of a rat. Thirty-five minutes after injection of calcium chloride or 40 min after injection of proline the rat was frozen in a mixture of hexane and solid carbon dioxide and blocked in 5% sodium carboxymethyl Cellulose. The carboxymethyl cellulose block was trimmed and a piece of copy paper was attached to the surface of the block with cellulose tape. Cryotome sections cut from the block were transferred from the paper to a glass slide coated with synthetic rubber adhesive. For wholebody autoradiography, sections were freeze-dried for 2 days and then placed against X-ray film. For light microscopic autoradiography, the freeze-dried sections were covered with a dried film of photographic emulsion. For histochemical use, the sections were fixed by raising the temperature to 4 C after immersion in 100% ethanol below -10 C. For histological observation, sections were postfixed with 2.5% glutaraldehyde and stained. Wholebody and light microscopic antoradiographs showed that sections so prepared could be used for the demonstration of soluble substances in wholebody sections and for detailed autoradiography at the light microscopic level, and the stained sections could be used for histological and histochemical studies.