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A method is described for the staining of glomerular basement membranes of aldehyde-fixed, osmicated renal tissue embedded in Epon or Araldite. The procedure presented does not require chemical digestion of the polymerized Epon or Araldite matrix before application of the methenamine-silver technique. There is no divergence from the procedures for fixation and embedding for transmission electron microscopy (TEM). This enables sections from the same block of renal tissue to be examined both by light microscopy (LM) and TEM without loss of time or sacrifice of quality.
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Scanning electron microscopy (SEM) is most often used to describe the structure of natural surfaces, but it can also be used to examine surfaces created artificially by sectioning or cryofracture. When plasma etched resin sections are studied, the SEM combines some of the functions of a light microscope and a transmission electron microscope. When etched frozen hydrated tissue is examined the groundwork is laid for the use of X-ray microanalysis for the study of elemental concentrations in tissue unexposed to chemical agents of any kind. Examples are given of the study of jejunum and stomach with these two techniques. The results are integrated with data from conventional SEM images to produce composite diagrams which assist with the interpretation of function and topographical morphology.
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A simplified method of low temperature methyl and butyl methacrylate embedding (up -20 degrees to -15 degrees C) is demonstrated using a proper redox system of benzoyl peroxide and aromatic amine. This method combines the morphological superiority of plastic-embedded bone tissue and bone marrow sections with the advantages of specific enzyme histochemical and immunochemical markers. The method permits good preservation of morphological details, the survival of antigenic determinants and the retention of enzyme activities. The specimens were fixed in 1.6% formaldehyde and 5% sucrose in 0.02 M phosphate buffer at pH 7.4, washed in 0.02 M phosphate buffer and 5% sucrose, dehydrated with acetone and impregnated with monomers of embedding medium. All these steps were carried out at +4 degrees C. The method presented is especially suitable for enzyme histological and immunohistological diagnosis of primary and secondary bone tumours, soft tissue tumours, as well as myelo- and lymphoproliferative disorders of bone marrow biopsies. Examples are demonstrated with mono- and polyclonal antibodies and reaction products of hydrolytic enzymes.
In the course of a chronic experimental study of polyarthritis in rats, we have established a plastic embedding technique which allows the processing not only of soft tissues but also of tissues such as cartilage, intraarticular ligaments, and even undecalcified whole rat joints and provides broad survival of enzymes and antigens. Preservation of morphological details and the precision of antigen localization are superior to those seen in cryostat and paraffin sections. The method seems to have much diagnostic and investigative value, especially in arthritis research.
Plastic embedding preserves tissue structure much more faithfully than does paraffin. Acrylic polymerization is innocuous to dye-binding groups in sections. The water solubility of glycol methacrylate monomer and the hydrophilic properties of the polymer allow for convenience in dehydration and for versatility in staining sections. Five years of experience with glycol methacrylate (GMA) embedding for light microscopy is summarized. Methods for purifying GMA monomer are cited. Procedures for fixing, dehydrating, embedding, polymerizing, sectioning and staining, using GMA, are explained. A method is provided for making glass knives long enough to cut large blocks. Simple, reliable, quick staining methods are outlined. When compared with paraffin, GMA offers opportunities for simpler, quicker procedures and yields sections of superior quality, greater information content, and less distortion.
As a macroscopic stain for gross brain sections to be embedded in plastic, tannic acid-iron alum is superior to the generally recommended LeMasurier's variation of the Berlin blue technique because of its greater permanency in plastic. However, as originally adopted for use with brain tissue by Mulligan, the intense black staining of gray matter is too dark for plastic embedded specimens. A modification of this method designed to overcome this difficulty is described. Staining procedure: Wash formalin-fixed brain slices overnight in running water. Wash in distilled water, 2 changes, 30 minutes each. Place slices individually in Mulligan's solution at a temperature of 60-65 C for 4 minutes. Rinse in ice water for 10 seconds. Mordant in 0.4% tannic acid in distilled water for 1 minute. Wash in running tap water for 1 minute. Develop in 0.08% ferric ammonium sulfate in distilled water until gray matter is light gray, about 10-15 seconds. Wash in lukewarm running water for 1 hour, then gently hand-rub whitish film from myelinated surfaces. Store briefly in 3% formalin or 25% glycerine if necessary depending on plastic embedding procedure to be followed.
Plastic embedding is a recently developed technique that has been shown to be superior to conventional paraffin embedding in the histopathologic identification of microorganisms in tissue specimens. This report describes a modification of the Brown and Hopps stain for the differentiation of gram-positive and gram-negative microorganisms in 3-micron-thick plastic sections. This technique is easily performed and has a rapid turnaround time. This staining method permits a more precise histopathologic diagnosis of infectious agents than is possible in paraffin-embedded tissues.
Ultrastructural immunocytochemical identification of transmitters in afferent terminals and targets of individual physiologically characterized neurons is essential for understanding the complex circuitry within the mammalian neocortex. For this type of analysis, we examined the utility of combining in vivo intracellular recording and biocytin injections with silver intensified 1 nm immunogold labeling of GABA and the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH). These transmitters are found to local neurons and afferents known to prominently modulate the activity of pyramidal neurons in the neocortex. Individual neurons were physiologically characterized and filled with biocytin in the frontal cortex of anesthetized rats. The brains were then preserved by vascular perfusion with aldehydes. Single vibratome sections through the recording site were reacted (1) for immunoperoxidase detection of biocytin and (2) for immunogold labeling of GABA or TH. Dually labeled sections were processed for light microscopy or embedded in plastic for electron microscopy. The dense peroxidase product for biocytin was detected in pyramidal neurons. These were located in superficial as well as deep cortical laminae, and were readily distinguished from immunogold silver labeling. GABA labeled terminals formed symmetric synapses with larger biocytin filled dendrites, whereas the TH labeled terminals contacted distal dendrites and spines. Peroxidase labeling for biocytin also was seen in a few axon terminals forming synapses with unlabeled and with GABA immunoreactive dendrites. These results suggest that single pyramidal neurons of the rat frontal cortex receive dual input from both GABA and catecholamine terminals. Additionally, this study demonstrates the usefulness of silver enhancement of 1 nm colloidal gold prior to plastic embedding for electron microscopic detection of neurotransmitters within afferents and targets of neurons physiologically characterized in vivo.
We describe a novel method that allows reliable detection of in situ hybridization signals in thin sections of plastic embedded embryos. Sections from plastic embedded embryos are thinner and have superior histological quality compared to paraffin, gelatin, agarose embedded sections or cryosections; however, plastic resin traditionally has not been used as an embedding medium following in situ hybridization because of loss of signal. When signal is detected with alkaline phosphatase and NBT/BCIP, the resulting colored precipitate is subject to fading when samples are exposed to organic compounds. The colored precipitate can be redeposited by repeating the NBT/BCIP reaction following plastic sectioning. This recolorization shows no loss of specificity, because signal is detected only where the anti-digoxigenin/alkaline phosphatase conjugated antibody is bound to the riboprobe. Strong signals can be detected without recolorization; however, weaker signals require the recolorization step. This novel method of re-depositing colored precipitate after processing and sectioning allows accurate determination of the location of gene expression and study of this expression in high quality histological sections of early chick embryos.
We describe modifications and improvements to our first report of a new nuclear emulsion coating technique for both light and electron microscopic autoradiography. Although this technique was originally designed to facilitate electron microscopic autoradiography, the methodology also allows pre-staining of plastic-embedded tissue sections prior to coating the slides with nuclear emulsion for light microscopic autoradiography. We now demonstrate that paraffin sections can be autoradiographically processed after being subjected to a combination of immunocytochemical reactions and special neuroanatomical strains. Parlodion film has been found to be more resistant to temperature changes and less prone to contamination than Formvar film. The shape of the double adhesive tape is an important aspect of the electron microscopic technique; it has been modified to minimize contamination and facilitate the removal of the grids from the glass slide. These technical adjustments facilitate the application of the nuclear emulsion and increase radionuclide specificity, thus expanding the investigative horizons of light and electron microscopic autoradiography.
Tartrate-resistant acid phosphatase (TRAP) has been proposed as a cytochemical marker for osteoclasts. We have developed an improved technique for the localization of TRAP in rat and mouse bone and cartilage. This procedure employs JB-4 plastic as the embedding medium, permits decalcification, and results in improved morphology compared with frozen sections. Peritoneal lavage cells were used to determine the appropriate isomer and concentration of tartrate necessary for inhibition of tartrate-sensitive acid phosphatase. After incubation in medium containing 50 mM L(+)-tartaric acid, osteoclasts and chondroclasts were heavily stained with reaction product. On the basis of their relative sensitivity to tartrate inhibition, three populations of mononuclear cells could also be distinguished. These three populations may represent: heavily stained osteoclast/chondroclast precursors; sparsely stained osteoblast-like cells lining the bone surface; and unstained cells of monocyte-macrophage lineage. Our results are consistent with the use of TRAP as a histochemical marker for study of the osteoclast.