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.     

Pyronin Y For Staining Stripped Autoradiographs Prepared from Plastic Embedded Sections of Rat Tissues Containing Plutonium

Autoradiography prepared by the stripping film technique from 1 μm sections of semithin plastic embedded liver and bone tissues were poststained for examination with a 1% pyronin Y solution. The use of this dye avoids heating the tissue section and the overlying photographic emulsion. It also allows the staining of the tissue section without excessive uptake of the stain by the gelatin of the stripping film.     

Stabilization of Fluorochromes After Immunofluorescent Staining of Tissue and Embedding in Epon

Tissue blocks or sections immunofluo-nt stnined before embedding, ig., liver and kidney, can be stored for more than 3 years without demonstrable fluorescence decay. The processing steps, including poststaining dehydration by alcohols and embedding in expoxy resins, seem to stabilize the fluorochromes fluo-in isotbiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) so that they fade I- during illumination. This is an advantage of the preembedding, immunofluo-nt staining technique which is combined with a lack of damage to the antigens by the plastic embedding medium.     

Silver Staining of the Nucleolar Organizer Regions (NORS) in Semithin Lowicryl Sections

The one-step silver technique was applied to semithin Lowicryl sections of root meristem cells of Allium cepa and a human tumor cell line (TG cells). In vegetal cells, after 5 min of staining reaction, the Ag-NOR proteins formed ring-shaped structures peripherally within the nucleolus. In animal cells silver granules were distributed over the entire nucleolus. The specificity of the staining reaction was increased by incubation of the sections in NH₄Cl and Schiff's reagent prior to Ag-NOR silver staining.     

Pararosaniline or acriflavine-Schiff staining of epoxy embedded tissue after periodic acid oxidation in ethanol: a method suitable for morphometric and fluorometric analysis of glycogen

A method for preparing tissue sections for automatic image analysis of glycogen is described. Large semithin sections of epoxy embedded tissue fixed in glutaraldehyde-osmium were stained with Schiff reagent and acriflavine (fluorescent staining) after resin removal and periodic acid oxidation in ethanol. We found it essential to avoid tissue rehydration before final staining. The Schiff stain permits an assessment of the cellular volume of glycogen, and the acriflavine allows a fluorometric evaluation of glycogen density.     

Cytochemical Staining of Sections from Plastic-Embedded Flagellates

Dinoflagellate chromosomes in sections of plastic-embedded cells were stained without removing the plastic. Azur B and Feulgen procedures were used to localise DNA. Azur B was used with Araldite or methacrylate sections by staining in 0.2% stain in 0.05 M citrate buffer at pH 4 for 1 hr at 50 C followed by rinsing in tertiary butyl alcohol to differentiate the chromosomes. Feulgen stain was used with Araldite sections by hydrolyzing in 1 N HCl at 60 C for 10 min, rinsing in water, staining for 24 hr, washing well, drying and covering. Fast green was used with methacrylate sections to stain proteins by flooding the slide with a 0.1% solution of stain in 0.06 M phosphate buffer at pH 8, allowing the stain to dry out at 40-50 C, washing well, drying and covering. Controls were carried out on material fixed in formalin and treated with nucleases or proteolytic enzymes prior to embedding, and staining.     

Pararosaniline Or Acriflavine-Schiff Staining of Epoxy Embedded Tissue After Periodic Acid Oxidation in Ethanol: A Method Suitable for Morphometric and Fluorometric Analysis of Glycogen

A method for preparing tissue sections for automatic image analysis of glyco-gen is described. Large semithin seaions of epoxy embedded tissue fixed in glutaraldehyde osmium were stained with Schiff reagent and acriflavine (fluorescent staining) after resin removal and periodic acid oxidation in ethanol. We found it essential to avoid tissue rehydra-tion before final staining. The Schiff stain permits an assessment of the cellular volume of glycogen, and the acriflavine allows a fluorometric evaluation of glycogen density.     

A Simple Technique for Osmicating and Flat Embedding Large Tissue Sections for Light and Electron Microscopy

Many investigators now use thin hand-sliced, tissue chopper, or Vibratome sections of fresh tissue in various procedures. In our experience brain and nerve sections varying in thickness from less than 40 to more than 300 μm, with or without prior embedding in agar, have a tendency to roll up or curl during aldehyde fixation and buffer washes. Once osmicated, such curled sections cannot be flattened. When the entire cut face of such thin slices is to be studied, sufficiently flat embedding so that some regions are not completely sectioned before others are even sampled is critical. This report describes fixation and flat embedding procedures, developed for light and electron microscopic autoradiographic studies of plastic embedded brain slices about 200 μm thick (Schwartz 1981), which can be applied to any comparable thin slice of nervous tissue (or potentially of many other tissues) to achieve maximally flat tissue faces. Since osmicated tissue slices are usually too thick to be transilluminated for direct examination with the light microscope, the methods described simplify preparation of the semithin sections required for this purpose.     

A Multichromatic Stain for Lowicryl K4M Embedded Tissues

A staining procedure using celes-tine blue and chrome-trope 2R for Lowicryl K4M embedded tissues is presented. The stain produces a reliable multichromatic stain for light microscopy on Lowicryl embedded semithin sections.     

New Rapid Methods for Tissue Diagnosis

Two new methods applicable to the staining of fixed and fresh frozen tissue sections are presented herein. In addition certain improvements are suggested for the technic reported by Geschickter, Walker, Hjort and Moulton (1931). In brief the procedures are as follows:

1. The thionin eosinate method of Geschickter et al (1931). This procedure has been modified as follows:
   1. A mixture of diethylene glycol, 40 parts, ethylene glycol, 40 parts, and grain alcohol, 20 parts is superior to ethylene glycol, 80 parts, and ethyl alcohol, 20 parts, as a solvent for the compound stain in that the staining is intensified.
   2. Ethylene glycol monobutyl ether supplants diethylene glycol monobutyl ether because of its lower viscosity.
   3. Ethyl phthalate replaces butyl phthalate on account of a more satisfactory viscosity.
2. The methyl green eosinate procedure is the same as the modified thionin eosinate method except for the following variations:
   1. The staining time is increased to one minute.
   2. Decolorization and washing are reduced to about 15 seconds.
3. The hematoxylin-eosin method. After cutting, the tissue sections are carried thru the following steps:

Unfold in water; transfer to formalin (4 to 40%) for at least 30 seconds; stain in hematoxylin (Harris) for 30 to 60 seconds; wash in water, 5 seconds; decolorize in 0.1% HC1 or saturated aqueous picric acid, 5 seconds; wash in water, S seconds; float in 0.5% ammonia, 5 to 10 seconds; wash in water, 5 seconds; stain in 5% aqueous eosin, 15 seconds¹; wash in water, 5 to 10 seconds; dehydrate in a mixture of diethylene glycol, 30 parts, and ethyl alcohol, 70 parts, 5 to 10 seconds; dehydrate in ethylene glycol monobutyl ether, 5 to 10 seconds; clear in ethyl phthalate, 5 to 10 seconds; float on a glass slide, blot with photographic lintless blotter, place a drop of neutral gum damar on the section, and cover with glass cover slip.     

Lamination of Thermoplastic Sheets as a Means of Mounting Histological Material

Permanent preparations of squashes, whole mounts and stained sections can be made by lamination of thermoplastic sheets. Classical procedures of staining and dehydration for sectioned material were used although dehydration after staining was not required for root tip squashes. Arranging the specimen with the identification label between two pieces of clear Vinylite plastic, 15 mils thick, tightening the preparation in a Photo-Seal Kit electric press and laminating for 3 min gave a finished preparation without the use of glass slides and cover slips. For root tip squashes, the stained tip was placed with a drop of stain between two pieces of plastic, squashed and then laminated. This insured retention of all the tissue which is sometimes lost during mounting processes. Preparations of unstained whole mounts were similarly laminated, with an identification label added between the plastic sheets. Stained sections were placed between two sheets of plastic but the identification label was placed on top of the preparation and a third piece of plastic added. This prevented the label from absorbing excess stain and the increased thickness allowed the slide to be used in a mechanical stage. Well preserved slides 18 mo old indicate that the laminated plastic slide is quite durable. It saves time, reveals good cytological detail and avoids some of the laborious features of other methods. It is a technic that can be used in introductory microtechnic and in the preparation of slides for class use in histology.     

Periodic acid incubation can replace hydrochloric acid hydrolysis and trypsin digestion in immunogold-silver staining of bromodeoxyuridine incorporation in plastic sections and allows the PAS reaction

Summary We have examined the possibility of improving the present methods of detecting bromodeoxyuridine (BrdU) and for combining the PAS reaction with the BrdU detection by means of immunogold-silver staining (IGSS). This was done in testes fixed in Carnoy or Bouin, and in parts of the small intestine which were fixed in Carnoy or periodate-lysine-paraformaldehyde (PLP). All tissues were embedded in a mixture of glycol methacrylate and butanediol-monocrylate. It was found to be impossible to carry out BrdU detection using HCl hydrolysis and trypsin digestion in combination with a PAS reaction. However, incubation of the plastic sections in periodic acid for a period of 30 minutes appeared to make it possible to eliminate the HCl denaturation step and to carry out a specific PAS reaction. Moreover, after incubation in periodic acid, trypsin digestion was no longer required to make the BrdU label accessible in GMA-embedded sections, nor to re-expose the antigenic sites in plastic sections of tissues fixed with cross-linking fixatives. In this way the loss of cell structures, which is inevitable when trypsin is used, can be avoided. Now a BrdU detection with improved morphology can be combined with the PAS reaction in the same plastic section in order to stain tissue carbohydrates. This is important for tumour diagnosis, where the PAS reaction can be very useful.     

An improved formulation of Nocht's azure-eosin stain for plastic embedded material

The popular azure-eosin stain of Nocht has been reformulated to provide consistent, intense staining of semithin sections of water-miscible methacrylate embedded tissues.     

An Improved Formulation of Nocht's Azure-Eosin Stain for Plastic Embedded Material

The popular azure-eosin stain of Nocht has been reformulated to provide consistent, intense staining of semithin sections of water-miscible methacrylate embedded tissues.     

Standardization of various applications of methacrylate embedding and silver methenamine for light and electron microscopy immunocytochemistry

The use of butyl-methyl-methacrylate embedding and the application of the silver methenamine (SM) method as a poststaining of the immunoperoxidase-DAB (IP) procedure led to the standardization of several useful methods for the visualization of tissue antigens at the light and electron microscope level. These procedures included: 1) Standardization of the actual methacrylate embedding; 2) The IP-SM method with and without periodic acid oxidation, which provided 100% intensification of the IP staining; 3) The IP-SM method made it possible to stain semithin sections (0.5 micron), and this in turn, permitted a) clear visualization under the light microscope of the intracellular distribution of antigens and, b) staining, in several adjacent sections, of roughly the same cytoplasmic region of the same cell with different primary antisera; 4) a double immunostaining whereby the first antigen in the sequence was revealed by the IP-SM method and the second by the IP procedure; 5) standardization of the IP and the IP-SM methods for post-embedding staining of ultrathin methacrylate sections. The combined application of methacrylate embedding and the IP-SM, and the use of an appropriate fixative, resulted in an ultrastructural immunocytochemical procedure characterized by a good immunoreactivity of the tissue sections, a strong and selective immunoreaction and a well preserved ultrastructure.     

Phosphotungstic Acid-Chromic Acid as a Selective Electron-Dense Stain for Plasma Membranes of Plant Cells

A mixture consisting of 1% phosphotungstic acid (PTA) in 10% chromic acid (CrO₃) selectively stains the plasma membrane of plant cells. Whole tissue or pelleted cell fractions are prepared for electron microscopy using conventional methods including glutaraldehyde fixation and OsO₄ postfixation, dehydration in acetone and embedding in Epon. To stain the plasma membrane, thin sections are transferred with a plastic loop to the surface of a 1% aqueous solution of periodic acid for 30 min for destaining. Following transfer through 5 distilled water rinses, the sections are exposed to the PTA-CrO₃ mixture for 5 min, rinsed and mounted on grids for viewing with the electron microscope. The selectivity of the stain is retained in homogenates and serves to identify the plant plasma membrane in cell fractions.     

A quantitative histochemistry technique for measuring regional distribution of acetylcholinesterase in the brain using digital scanning densitometry

Studies of brain acetylcholinesterase (AChE) are traditionally based on biochemical assays, immunoreactivity, and histochemistry. Conventional histochemistry yields rich morphological data from tissue sections but yields quantitative results only with great difficulty. Several histochemical methods developed in recent years, including microdensitometry, microphotometry, and video-based histochemistry, are effective in quantitative and detailed study of AChE in tissue sections. However, they are usually time-consuming. As we report here, we adapted digital scanning densitometry to quantitate AChE histochemical staining in brain sections. The AChE and butyrylcholinesterase (BuChE), as measured by the method, were heterogeneously distributed throughout the brain, results that are consistent with those obtained by biochemical methods. The staining intensity is dependent on section thickness, substrate concentration, and reaction time. The cholinesterase inhibitor methyl paraoxon significantly decreased AChE staining intensity. Furthermore, data acquired from densitometry are similar to those obtained by video-based microscopy or by spectrophotometry. The advantage of the densitometric measurements compared to other quantitative histochemical methods is that it is very rapid while collecting data that are equivalent in quality. Because the digital scanning densitometers provide high quality and sensitive imaging, wide dynamic ranges, and convenient image analysis software, they are very useful tools in quantitative histochemistry.     

A rapid method for staining semithin tissue sections embedded in araldite

A new rapid method is proposed for staining of semithin sections. The method involves the treatment of sections with a methylene blue solution with a slight heating for 1-3 minutes. The method allows to receive polychromatic contrast preparations, to save time and reagents. It also permits to avoid restaining effect. The preparations can be preserved for a long time in plastic media (e.g. in polystyrene) for light microscopy. A comparative analysis of the staining methods used in the electron microscopic practice is given.     

Staining and Histomorphometry of Microcracks in the Human Femoral Head

We developed staining techniques that permit identification and histomorphometric analysis of microcracks in the human femoral head 1) from thick, ground bone sections (100 μm) by prestaining with the Villanueva mineralized bone stain (MIBS), and 2) from plastic embedded, undecalcified thin bone sections (5-15 μm) by staining in gallocyanin chrome alum-Villanueva blood stain methods. Both methods represent a significant improvement in the stainability of the microcracks, cellular and tissue elements, and the simultaneous assessment of osteoid seams and tetracycline markers by histomorphometry. Shrinkage and other artifacts were minimized, which helped to clarify some of the uncertainties arising from artifacts resulting from some bone staining methods. Histomorphometric analyses of microcracks were conducted on thick, ground sections of subchondral and trabecular bone. Microcracks were more prevalent in the subchondral bone and osteochondral junction than in the more distant trabeculae. We have consistently localized microcrack areas in bone tissues prepared in these ways.