A new integrated concept for the improved preparation of sections of fresh or frozen tissue for light microscope histochemistry

Summary In order to obtain thin sections of plant tissues which combined good morphological preservation and the preservation of the substances and enzyme activities in the tissues, a concept of section preparation by external stabilization was developed. The main components are as follows: (1) appropriate supporting medium; (2) surface coating before each sectioning process, the coating being either non-permanent, permanent, or semi-permanent; (3) suitable techniques for affixing the coated sections to the slides using either pressure-sensitive adhesive or solvent-based adhesive; and (4) mounting media with defined refractive indices (preferably UV-curable, water-soluble monomers). By this approach, sections exhibiting excellent morphological and physiological preservation were obtained using either a cryostat at –30°C or a rotary microtome at room temperature.     

Electron microscopic dry-mounting radioautography for diffusible compounds by means of ultracryotomy

Summary Cultured HeLa cells or mouse liver and pancreas tissues were labeled with ³H-thymidine, -uridine or -glycine for varying periods in vitro, frozen in liquid nitrogen and cut on an LKB ultrotome equipped with LKB Cryokit. Dry ultrathin sections were mounted on grid meshes and were either air-dried, freeze-substituted or freeze-dried, and were covered with dry films of radioautographic emulsions, exposed, developed, stained and were observed in electron microscopes.After a number of trials, it was possible to obtain fairly good preservation of both cell structure and radioisotopes by means of freeze-dried and drymounted ultrathin frozen sections. However, the results are not completely satisfactory at the present time.The outline of this paper was presented at the Symposium on Radioautography at the 5th International Congress of Histochemistry and Cytochemistry held in Bucharest, Romania, 30 August–2 September 1976     

Decreased D2-40 immunoreactivity in stored paraffin sections and methods for preserving it

D2-40, a monoclonal antibody against podoplanin, is a selective marker of lymphatic endothelium and is widely used for research on and diagnosis of pathology of lymphatic vessels. We examined the relation between the duration of tissue section storage and changes in immunostaining by D2-40 antibody; we evaluated also the effects of preservation methods on changes in immunostaining during storage. Staining by D2-40 was attenuated by long-term preservation of scalp skin and lymph node sections at room temperature. The attenuation of D2-40 staining in stored sections was improved by preservation at low temperature, i.e., 4° or ? 30° C. We investigated also the immunostaining of preserved tissue sections using NZ-1 and Lyve-1, which are antibodies against lymphatic endothelium markers. Staining by NZ-1 or Lyve-1 antibody was detected clearly in sections that had been stored for 16 weeks. Our study suggests that either long-term storage of D2-40 immunostained tissue sections should be avoided or the section should be preserved at low temperature.     

Vacuum Freeze Drying of Frozen Sections for Dry-Mounting, High-Resolution Autoradiography

Specimens no larger than 1.5 × 1.5 × 2 mm were frozen in liquid nitrogen and sectioned, while still frozen, with a refrigerated microtome. The frozen sections were dried in a vacuum, then pressed onto either Kodak NTB10 plates or onto slides which had been coated with Kodak NTB3 emulsion and dried. Radioactive mouse liver was used to test tissue preservation. Intestinal mucosa with Ha-labeled nuclei was used to test the quality of autoradiography. Good cytological detail was preserved in both tissues, with the autoradiographs interpretable at the cellular level.     

An improved method for preparing cryostat sections of undecalcified bone for multiple uses.

We developed a technique that permits the use of serial sections (7-20 microns) from a single fixed piece of bone tissue for immunofluorescence, measurement of fluorescent bone labels, enzyme histochemistry, and general staining. This technique combines modifications of previously established methods with perfusion of the polymer polyvinylpyrrollidone (PVP) to improve sectioning, and produces reliable sections with good preservation of both hard and soft tissues. The combination of techniques from several workers, the use of perfusion with a polymer to increase the sectionability of the bone, and the addition of a gelatin adhesive on top of pressure-sensitive adhesives represent a significant improvement over previously described methods. The sections obtained are usable for immunocytochemistry, general staining, enzyme histochemistry, and visualization of fluorescent bone labels. We have consistently used tissues prepared in this manner for immunohistochemical demonstration of neuropeptides in skeletal tissues and for localizing tartrate-resistant acid phosphatase (TRAP). In addition, other tissues obtained from PVP-perfused rats, such as brain, spinal cord, muscle, gut, and sympathetic ganglia, are also well preserved and demonstrate immunohistochemical staining comparable to and possibly superior to that obtained with normal fixation protocols.     

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.     

Unconventional application of standard light and electron immunocytochemical analysis to aldehyde-fixed, araldite-embedded tissues

A simple procedure for the immunocytochemical analysis of glutaraldehyde/formaldehyde-fixed, Araldite- or Epon-embedded tissues by either light or electron microscopy is presented. Retention of immunoreactive antigen in deplasticized sections was achieved by use of a low concentration of glutaraldehyde in the fixative in combination with a seldom-used plastic solvent. This protocol produced good ultrastructural preservation in tissues and large, high-quality, 2-micrometers thick, plastic-free sections. These semithin sections provided a level of structural and antigenic preservation, image resolution, and labeling intensity that surpassed all other conventional sectioning methods used for immunocytochemistry. The capacity to use a single tissue sample in studies designed for light and electron immunocytochemistry, in conjunction with existing autoradiographic and cytochemical techniques, makes this a very desirable method for routine tissue preparation in research and clinical applications.     

A new method for transfer of polyethylene glycol-embedded tissue sections to silanated slides for immunocytochemistry

Polyethylene glycol (PEG) is an excellent embedding medium for immunohistochemical studies. It provides structural preservation superior to frozen sections and increased sensitivity of antigen detection compared with paraffin sections. One limitation of PEG embedment is that PEG sections are difficult to handle and adhere poorly to glass slides. Here we present a simple and effective method for embedding tissues in PEG and transferring the resultant sections onto silanated glass slides. In addition, a method for silver enhanced colloidal gold immunostaining was combined with common dye staining to demonstrate the excellent structure preservation and sensitive antigen detection. Bovine chorionic membrane was fixed with Bouin's fixative, embedded in polyethylene glycol (PEG) 1500, cut into 5-microns sections, flattened over agarose blocks (10 x 10 x 2 mm3), and blotted onto Digene silanated slides. Slides were then washed in PBS, which removed the PEG and agarose blocks. Tissue sections were immunocytochemically stained with dilute antiserum raised in a rabbit against purified bovine placental retinol binding protein (bpRBP). Sections were washed and incubated with 1-nm colloidal gold-labeled goat anti-rabbit IgG. The immunogold particles were enhanced by silver staining (IGSS). Specimens were observed and photographed with an Olympus epipolarization microscope. The new method offered excellent morphological preservation of cell structure and the epipolarization microscopy provided high sensitivity for detection of specific immunogold-silver particles.     

Polyacrylamide gel electrophoresis and immunoblotting of proteins extracted from paraffin-embedded tissue sections

We show in this communication that polyacrylamide gel electrophoresis (PAGE) and immunoblotting of proteins can be performed using one to two 5-7 micron paraffin sections of tissues fixed in non-cross-linking fixatives (acetone, alcohol, or modified Carnoy's solution). Proteins for study were extracted from paraffin sections of mouse foot pad and liver. The presence of unaltered keratin polypeptides in tissues fixed with either acetone or alcohol was demonstrated in gels stained with Coomassie brilliant blue. The preservation of their antigenic determinants was demonstrated with immunoblotting. Furthermore, the immunoreactivity of soluble proteins, such as albumin, remained unaltered in immunoblots obtained from paraffin-embedded mouse liver sections. These data indicate that tissues embedded and stored in paraffin are useful for the above-mentioned biochemical and immunological studies and may therefore be an important technique for diagnostic pathology or retrospective studies.     

Immunogold labeling of luciferase in the luminous bacterium Vibrio harveyi after fast-freeze fixation and different freeze-substitution and embedding procedures

We studied the ultrastructural localization of luciferase on sections of the bioluminescent bacterium Vibrio harveyi by indirect immunogold staining, using a polyclonal antiluciferase antibody and the usual control tests, after chemical fixation or fast-freeze fixation (FFF) followed by different freeze-substitution (FS) procedures and embedding in either Epon or LR White. After liquid fixation with glutaraldehyde and paraformaldehyde and LR White embedding, labeling occurred over the cytoplasm but not over the condensed nucleoid. Epon embedding almost abolished it. FFF-FS considerably improved the morphological preservation and revealed cytoplasmic "patches" with a complex ultrastructure in Epon sections. The preservation was always less good in LR White. The patches were densely labeled, even in Epon sections, after FS in acetone. However, labeling intensity was 3.7 times greater in LR White than in Epon. With both resins, labeling diminished similarly when fixative agents were present in the FS medium. The localization of luciferase in the cytoplasm and particularly in the patches is discussed.     

Radioautography of Histologic Sections of Human Testicular Tissue

Two techniques suitable for qualitative radioautographic analysis of histologic sections of human testicular tissue are described. In one, deparaffinized wet slides are coated with photographic emulsion resulting in significant improvement in preservation of histologic quality of the tissue section. In the second procedure stained and processed slides are coated with a thin layer of H.S.R. prior to coating with photographic emulsion. This procedure allows excellent preservation of the stained tissue section and also enables visualization of the reactive cell and its radioautographic image at two different focal planes. The techniques can be applied with equal advantage to histologic preparations of other tissues.     

A solvent-free coating-procedure for the improved preparation of cryostat sections in light microscope histochemistry

Summary A new technique is presented for the external stabilization of cryostat sections by spraying the specimen surfaces with an aqueous solution of poly(vinyl alcohol) before each sectioning stroke. The spray freezes upon the surface and forms a tough coating which facilitates subsequent sectioning and handling especially of difficult material. The sections are affixed upon cold glass slides covered with an improved formulation of pressure-sensitive adhesive. During further processing of the affixed sections, the PVA-coating and any surrounding supporting medium dissolve without traces in the first aqueous incubation or staining solution.     

Postembedding immunohistochemical demonstration of antigen in experimental polyarthritis using plastic embedded whole joints

Summary A method is presented for the immunohistochemical demonstration of antigens in whole undecalcified joints of small laboratory animals. With this method of tissue preparation, involving embedding in a medium mainly based on 2-hydroxyethyl methacrylate, preservation of antigenicity is satisfactory. Antigens can be demonstrated in 2 m sections by either immunofluorescence or immunoperoxidase and an indirect technique. Therefore in addition to the morphological analysis of joint alterations in experimental polyarthritis, there is now an opportunity to trace the inciting antigen and to study in parallel the enzymatic equipment of the cells involved, using consecutive sections from a single block of tissue.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 54, C2     

Ultrastructural localization of nucleic acids through several cytochemical techniques on osmium-fixed tissues: comparative evaluation of the different labelings

Several cytochemical techniques, such as sodium tungstate, acid hydrolysis phosphotungstic acid (HAPTA), ethylenediaminetetraacetic acid (EDTA), RNase-gold, and osmium-ammine, have been applied for the ultrastructural demonstration of nucleic acids on sections of tissues fixed in glutaraldehyde postfixed with osmium tetroxide and embedded in Epon. In order to obtain specific results, the sections had to be treated with sodium metaperiodate prior to performing the labeling protocol. The results for each method were identical to those obtained on nonosmicated tissues; the main difference being the enhancement in the ultrastructural preservation, which allowed for higher resolution. In addition to these techniques, and for comparative evaluations, DNA was also revealed by the DNase-gold approach on nonosmicated tissue sections. The consistency in the results, obtained over the nucleus with either EDTA or the RNase-gold complex for revealing RNA and those obtained with either osmium-ammine or DNase-gold for revealing DNA, supports the high specificity of the RNase-gold, DNase-gold, and osmium-ammine techniques. Furthermore, these results demonstrate the possibility of performing various cytochemical techniques on tissues processed for routine electron microscopy.     

In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling.     

Painting and printing living bacteria: engineering nanoporous biocatalytic coatings to preserve microbial viability and intensify reactivity

Latex biocatalytic coatings containing approximately 50% by volume of microorganisms stabilize, concentrate and preserve cell viability on surfaces at ambient temperature. Coatings can be formed on a variety of surfaces, delaminated to generate stand-alone membranes or formulated as reactive inks for piezoelectric deposition of viable microbes. As the latex emulsion dries, cell preservation by partial desiccation occurs simultaneously with the formation of pores and adhesion to the substrate. The result is living cells permanently entrapped, surrounded by nanopores generated by partially coalesced polymer particles. Nanoporosity is essential for preserving microbial viability and coating reactivity. Cryo-SEM methods have been developed to visualize hydrated coating microstructure, confocal microscopy and dispersible coating methods have been developed to quantify the activity of the entrapped cells, and FTIR methods are being developed to determine the structure of vitrified biomolecules within and surrounding the cells in dry coatings. Coating microstructure, stability and reactivity are investigated using small patch or strip coatings where bacteria are concentrated 102- to 103-fold in 5-75 microm thick layers with pores formed by carbohydrate porogens. The carbohydrate porogens also function as osmoprotectants and are postulated to preserve microbial viability by formation of glasses inside the microbes during coat drying; however, the molecular mechanism of cell preservation by latex coatings is not known. Emerging applications include coatings for multistep oxidations, photoreactive coatings, stabilization of hyperthermophiles, environmental biosensors, microbial fuel cells, as reaction zones in microfluidic devices, or as very high intensity (>100 g.L-1 coating volume.h-1) industrial or environmental biocatalysts. We anticipate expanded use of nanoporous adhesive coatings for prokaryotic and eukaryotic cell preservation at ambient temperature and the design of highly reactive "living" paints and inks.     

The morphological and immunohistochemical analysis of renal biopsies by light and electron microscopy using a single processing method

A methodology is described in which a number of well-established research techniques are brought together to enable the complete diagnostic analysis of a renal biopsy on a single piece of tissue. By embedding the biopsy in the acrylic resin LR White, unsupported sections of which are stable in the electron beam, light and electron microscopy and immunocytochemistry become feasible on sections from the same block. The biopsy is glutaraldehyde fixed but post-fixation in osmium tetroxide, which is often deleterious to antigen preservation, is omitted. Extraction in organic solvents and resin monomer is minimized by rapidly infiltrating the tissue from 70% ethanol and polymerizing the resin catalytically at 0 degrees C. Semithin sections can be stained with haematoxylin and eosin, Toluidine Blue or methenamine silver, giving results similar or superior to those obtained from paraffin sections. Thin sections show that the standard of morphological preservation is similar to that seen using epoxide sections even though the kidney is unosmicated. The tissue retains a high level of antigen reactivity, which, in the limited number of cases so far examined, has paralleled or exceeded that demonstrated by conventional immunofluorescence on frozen sections.     

Immunocytochemical localization of nerve growth factor: effects of fixation

The fixation dependence of immunocytochemically demonstrable nerve growth factor (NGF) was investigated. Several commonly used fixation methods have been employed, including buffered formaldehyde, Bouin's fluid, and chloroform-methanol, as well as freezing and cryostat sectioning. The immunostaining technique was an immunoenzyme bridge procedure on either paraffin sections or frozen sections. Of those methods tested, fixation for 1 hr in a buffered formaldehyde appeared to provide optimal preservation and localization of immunoreactive material. Using this method, reaction product was localized in granules of the granular tubule cells of the male mouse submandibular gland. Prolonged fixation in buffered formaldehyde resulted in a diffuse background staining and loss of granule immunoreactivity. In frozen sections and in tissues fixed with either Bouin's solution, chloroform-methanol, or buffered paraformaldehyde-glutaraldehyde increased cytoplasmic background staining and loss of granule immunoreactivity were observed. It was concluded that, for the localization of NGF at the light microscopic level, a brief (1 hr) buffered formaldehyde fixation is optimal.     

Histological techniques and microscopic analysis of biological agents for preservation of human bone remains

Microscopy and histology techniques can be applied to morphological study of fungi and bacteria contaminating ancient human osteological remains. Undecalcified samples are cut with a diamond rotary blade microtome and an original technique was applied using adhesive tape to prevent damage to the bone surface during sectioning. We used light microscopy, polarized light microscopy, and epi-illumination fluorescence systems. Common dyes can be applied to 80 μm sections using classic staining techniques to reveal the architecture of bone and the presence of infecting biological agents.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8–12 h at –50 to –40°C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45°C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4°C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.