Contraction-Free, Fume-Fixed Longitudinal Sections of Fresh Frozen Muscle

Contraction damage occurring when longitudinal frozen sections of fresh unfixed muscles are thawed on microscope slides has limited histological examination of this tissue mainly to cross sections. Longitudinally oriented sections are advantageous for investigating properties that vary along the length of the muscle fibers. A fume fixation technique has been developed for preventing contraction of thick longitudinal frozen aections. The technique is compatible with histochemical staining of enzymes.     

The use of cryomethods for research on the sarcomere ultrastructure of rabbit skeletal muscles

The ultrastructure of sarcomeres of glycerinated rabbit psoas muscle was studied using freeze-fracture-etching, freeze-drying and optical diffraction techniques in comparison with the investigation of this muscle by plastic sections and negative staining methods. In frozen and dried myofibrils isolated from the above muscle the stripes of minor proteins location in A- and I-disks were clearly seen. The pivot structure in thick filaments was revealed in longitudinal fractures of the muscle. The ordered arrangement of myosin heads (crossbridges) associated with actin filaments was preserved in frozen longitudinal fractures as evidenced by optical diffraction. Freeze etching technique allowed to revealed some details of Z-line structure: alpha-actinin bridges connecting the ends of actin filaments of neighbouring sarcomeres and to preserve the lateral struts between actin filaments in I-disks.     

The birth of mice from testicular spermatozoa retrieved from frozen testicular sections

Male gamete cryopreservation has been widely used for both human reproduction and animal breeding. We investigated whether testicular spermatozoa retrieved from frozen testicular sections (10 or 25 mum thick) could support the full-term development of normal progeny. For this purpose, frozen testicular sections were prepared from two genetic backgrounds (BDF1 or B6 GFP transgenic mice), and the functional ability of testicular spermatozoa after preservation for 1 day, 1 mo, and 3 mo was assessed by intracytoplasmic sperm injection (ICSI). Testicular spermatozoa were successfully retrieved from frozen testicular sections for the use of ICSI, regardless of the preservation period. The ICSI technique revealed that oocytes (BDF1 or B6 background) injected with testicular spermatozoa prepared from frozen testicular sections developed into normal progeny, even though the sections had been cryopreserved for 3 mo at -30 degrees C. Approximately 15% and 5% of the embryos preserved for 3 mo developed to full term if the testicular spermatozoa were prepared from the 25- and 10-mum sections, respectively. These results clearly indicate that male gametes can be viably preserved in frozen testicular sections. The technique described herein will allow the preservation of male gametes in the form of a "book" or "file" by mounting the sections on a paper-thin sheet. Furthermore, this technique may be of value in the clinical treatment of severe male infertility, since testicular spermatozoa can easily be found through examination of testicular cross sections rather than by attempts to identify them in testicular cell suspension.     

Histological Methods for Assessing Myelin Sheaths and Axons in Human Nerve Trunks

Although there are many histological techniques for assessing myelin sheaths and axons in paraffin embedded or frozen sections of the peripheral nervous system, modern approaches usually use plastic embedded material. Although plastic embedding is superior for small cutaneous branches, this method has limited value for histological assessment of nerve trunks. We report three methods which together yield a comprehensive approach for thorough and detailed investigation of human nerve trunks. The rapid osmication method permitted assessment of myelinated nerve fibers from frozen sections at operation, thus providing the surgeon with guidance on the extent of nerve resection. The modification presented here resulted in permanent slides, allowing comparison of results with those of the other two procedures. The new osmium-hematoxylin technique could be performed on paraffin embedded nerves. Paraffin, unlike plastic, permitted the study of the whole cross sectional area of the nerve in single sections. Moreover, the sharp image of the myelin permitted computerized morphometry. The significantly modified axonal silver impregnation technique was performed on frozen sections mounted on glass slides, as opposed to the time-consuming impregnation of free-floating sections. The latter technique had a high success rate and permitted semiquantitative assessment of axons in nerve trunks. These methods can be performed in any routine histology laboratory and resulted in greater accuracy compared to conventional methods.     

A rapid, sensitive histochemical stain for myelin in frozen brain sections.

A staining technique is described whereby frozen brain sections are incubated in a vehicle containing gold chloride. After 2-4 hr in this solution, both large myelinated bundles and fine individually myelinated fibers are darkly stained. Advantages of this technique over conventional myelin stains include speed, sensitivity, metachromatic staining, and compatibility with formalin-fixed and frozen cut sections. Possible histochemical mechanisms are discussed.     

Improved method for making high-affinity sections of soft tissue embedded in polyethylene glycol (PEG): its use in screening monoclonal antibodies

This article describes improvements in the immunohistologic technique for embedding highly hydrated embryonic tissue in polyethylene glycol 1000 (PEG)--a water-soluble wax of melting point 39 degrees C--and compares the PEG sections with frozen and polyester-wax sections. The main improvement ensures that relatively large PEG sections (8 X 3 mm) stretch out and adhere well to slides: a coat of albumen and glycerine is dried onto the slides and a fresh coat applied just before use. The embedding, sectioning, and mounting procedures, which are considerably faster than those for wax processing, have been developed for screening monoclonal antibodies against the differentiated neural crest cells in the anterior eyes of 9-day-old chick embryos. PEG sections of such eyes were a little fragile, but showed good cellular detail, similar to or better than in wax sections and considerably better than in frozen sections. The responses of PEG sections to the antibodies were far stronger than those of wax and marginally better than those of frozen sections. In one experiment using 125I-labeled rabbit anti-mouse antibody on sections previously treated with antibodies or antisera, PEG sections bound about five times as much label as wax sections and approximately 30% more than frozen sections. The main limitation of the technique is that, because of the softness of PEG, it only works well for embedding a limited range of tissues. Such PEG sections may, however, be useful for in situ hybridization as well as for immunohistochemistry.     

A Hydraulic Powered Microtome in a Commercial Freezer; An Improved Cryostat for Large Sections

Modifications have been made on the Ullberg technique of taking whole-body sagittal sections of frozen mice on Scotch tape. Three improvements are described which greatly increase the ease and reliability of taking the sections. The microtome is driven by a hydraulic system for a smooth, dependable stroke. The microtome stage has been redesigned to eliminate uneven sections. The cryostat is an ordinary, commercial freezer of the frost-free design which eliminates the need for defrosting and also maintains a lower humidity.     

A combined silver and acetylcholinesterase method for staining intramuscular innervation.

A combined acetylcholinesterase and silver stain for demonstrating the intramuscular innervation of fresh frozen tissue is described. Intramuscular nerves, subterminal axons, and motor end plates are simultaneously stained brown or black with minimal staining of connective tissue and muscle fibers in longitudinal sections 30-100 mu thick. The method has been applied to fetal and adult rat, porcine, and bovine skeletal muscle. Antemortem and postmortem tissue samples stained equally well. The method facilitates simultaneous appreciation of morphological alterations in nervous and muscular tissues; in clinical and research laboratories alike it is of value when muscle abnormalities which may be related to disorders of nervous origin are studied. Compared with other published procedures this method has shorter time requirements, uses fresh frozen tissue, and displays superior staining characteristics.     

A Combined Silver and Acetylcholinesterase Method for Staining Intramuscular Innervation

A combined acetylcholinesterase and silver stain for demonstrating the intramuscular innervation of fresh frozen tissue is described. Intramuscular nerves, subterminal axons, and motor end plates are simultaneously stained brown or black with minimal staining of connective tissue and muscle fibers in longitudinal sections 30-100 μ thick. The method has been applied to fetal and adult rat, porcine, and bovine skeletal muscle. Antemortem and postmortem tissue samples stained equally well. The method facilitates simultaneous appreciation of morphological alterations in nervous and muscular tissues; in clinical and research laboratories alike it is of value when muscle abnormalities which may be dated to disorders of nervous origin are studied. Compared with other published procedures this method has shorter time requirements, uses fresh frozen tissue, and displays superior staining characteristics.     

ELECTRON PROBE MICROANALYSIS OF SILICON DEPOSITION IN THE INFLORESCENCE BRACTS OF THE RICE PLANT (ORYZA SATIVA)

The deposition of silicon in tissues of the inflorescence bracts of rice has been studied with the electron probe microanalyzer. Tissues for analysis were prepared by means of peels, frozen transverse and longitudinal sections, chromations and macerations. The microanalysis shows the heaviest deposition in a layer external to the abaxial (outer) epidermis. The cells of this epidermis are only sparsely silicified, but the’ imprints of these cells are left on the outer silica layer. In the inner tissues of the bracts, silicon deposition is mostly associated with the cell walls.     

A Bodian method for mounted frozen sections.

For application of the Bodian method to frozen sections, cut frozen peripheral nerve or muscle at 10 mum and mount. Fix for 4 days in 18 parts 80% ethanol, 1 part 10% formalin, and 1 part glacial acetic acid. Fix central nervous tissue in the same mixture prior to freezing and sectioning, and after mounting postfix for 4 days. Impregnate by the Bodian procedure. The results equal Bodian stains of paraffin sections. The technique is simple and reliable. The use of 10 mum frozen sections produces little artifact and allows alternate serial sections to be stained with other techniques.     

Discrimination Between Paraffin-Embedded and Frozen Skin Sections Using Synchrotron Infrared Microspectroscopy

The difference between paraffin-embedded and frozen skin sections is always questionable. Ten patients of early stage mycosis fungoides, ten patients with psoriasis and ten normal controls were included in this study. Aim of this study is to differentiate between paraffin-embedded and frozen skin sections in inflammatory and malignant dermatoses using synchrotron infrared microspectroscopy (SIRM). It was found that epidermal beta sheets in paraffin-embedded sections were higher in a highly significant manner than frozen sections (P < 0.001). Also, epidermal nucleic acids in paraffin-embedded sections were lower in a highly significant manner than frozen sections (P < 0.001). However, when various skin diseases were compared with the control. It was found that the difference between paraffin-embedded and frozen skin sections were almost similar. In conclusion SIRM is a unique promising diagnostic technique and it seems that frozen processing preserve skin tissue more, this was represented by less apoptosis (beta sheets) and more nucleic acids than paraffin processing. However, there are still many advantages of both approaches over the other depending on the goal of the study.     

A TECHNIQUE FOR ULTRACRYOTOMY OF CELL SUSPENSIONS AND TISSUES

Ultracryotomy of fixed tissue has been investigated for a number of years but, so far, success has been limited for several reasons. The simple technique herein reported allows the ultracryotomy not only of a variety of tissues but also of single cells in suspension, with a preservation and visualization of ultrastructural detail at least equivalent to that obtained with conventional embedding procedures. In this technique, sucrose is infused into glutaraldehyde-fixed tissue pieces before freezing for the purpose of controlling the sectioning consistency. By choosing the proper combinations of sucrose concentration and sectioning temperature, a wide variety of tissues can be smoothly sectioned. Isolated cells, suspended in a sucrose solution, are sectioned by sectioning the frozen droplet of the suspension. A small liquid droplet of a saturated or near-saturated sucrose solution, suspended on the tip of an eyelash probe, is used to transfer frozen sections from the knife edge onto a grid substrate or a water surface. Upon melting of the sections on the surface of the sucrose droplet, they are spread flat and smooth due to surface tension. When the section of a suspension of single cells melts, individual sections of cells remain confined to the small area of the droplet surface. These devices make it possible to cut wide dry sections, and to avoid flotation on dimethyl sulfoxide solutions. With appropriate staining procedures, well-preserved ultrastructural detail can be observed. The technique is illustrated with a number of tissue preparations and with suspensions of erythrocytes and bacterial cells.     

Rapid Nissl Staining for Frozen Sections of Fresh Brain

Working with X-ray film autoradiography of soluble isotopes, we needed a staining technique for the localization of nuclei in frozen sections of fresh brain. We have found no Nissl staining method in the literature concerning autoradiography specially recommended for this purpose, nor have we found in handbooks on staining a Nissl method clearly recommended for unfixed, frozen sections of brain. The methods described are intended for paraffin or celloidin sections, and require fixation of brain before sectioning (which must be avoided when working with soluble isotopes). Because autoradiography is a time-consuming method, any technique which shortens time needed for the overall procedure is welcome. Most Nissl techniques described in the literature require long preprocessing of the tissue. We found two rapid methods, described by Humason (1967) and LaBossiere and Glickstein (1976), but their application to frozen sections did not give good results. After trials with several types of techniques, we succeeded in developing two Nissl modifications with slightly different qualities, one of 12 min and the other of 2-3 h. The longer method includes conventional steps in staining; the shorter method does not include fixation or lipid extraction. These methods were applied to 20-60 μm brain sections cut in the cryostat at -10 to -12 C and dried on gelatinized slides.     

A histochemical study of adenosine triphosphatase and other nucleotide phosphatases in young root tips

Summary The distribution of ATP-ase and other nucleotide phosphatases has been studied in young root tips of maize, barley and broad bean using frozen and paraffin sections stained by standard lead sulphide precipitation procedures. High ATP-ase activity was found at the root and cell surface which is in agreement with previous biochemical studies using excised roots and cell wall preparations. Staining was also found in the nuclei and at particulate sites in the cytoplasm. Differences were observed between the present work and the staining pattern obtained for -glycerophosphatase, and between ATP-ase staining in the three roots studied. These results are discussed in relation to the possible physiological activity of the enzymes and to the differences found between earlier histochemical studies of ATP-ase activity.Abbreviations F.L. frozen longitudinal - F.T. frozen transverse     

On the preparation of cryosections for immunocytochemistry

The key preparation steps in the Tokuyasu thawed frozen section technique for immunocytochemistry, namely freezing, sectioning, thawing, and drying, were studied. A spherical tissue culture cell was used as a model system. The frozen hydrated section technique indicated that glutaraldehyde-fixed, 2.1 M sucrose-infused pellets of cells were routinely vitrified by immersion in liquid nitrogen but water was crystallized when lower sucrose concentrations (0.6-1 M) were used. Quantitative mass measurements showed that the fixed cells are freely permeable to sucrose. The frozen hydrated sections were severely compressed but cell profiles regained their circular appearance upon thawing. The average section thickness of our frozen-hydrated sections was 110 nm: this was reduced to 30-50 nm upon thawing, washing, and air-drying. This change was accompanied by severe drying artifacts. By using the methyl cellulose drying technique, this collapse upon air-drying could be significantly reduced, but not completely prevented, giving an average thickness of 70 nm.     

A Method for the Histochemical Differentiation of Cholesterol and Its Esters

Free cholesterol is demonstrated in formalin-fixed frozen sections when treated successively by digitonin, alcohol-ether, and the Schultz technique, in which circumstances cholesterol esters are not visualized. Cholesterol esters and free cholesterol are both demonstrated in comparable sections treated by the Schultz method alone, so that the difference between such sections indicates the sites at which cholesterol esters may be considered present.     

Evidence of Transcellular Strands in Transverse Cryostat Sections of Cucurbita pepo Sieve Tubes

Transverse cryostat sections of rapidly frozen vascular bundlesof Cucurbita pepo were viewed in a microscope with Nomarskioptics. Structural bodies were frequently observed in sievetube lumina and filling the sieve plate pores. The bodies consistof an outer boundary ring and an inner core, often granularin appearance, and may represent transverse sections of boundedtubes filled by substructural material. This evidence is consistentwith earlier observations of strand-like structures with parallelsubstructural elements seen in longitudinal sections.     

A coupled peroxidatic oxidation technique for the histochemical localization of monoamine oxidase A and B and benzylamine oxidase

Summary A coupled peroxidatic oxidation technique is presented which employs benzylamine and tyramine as substrates and clorgyline, deprenyl, phenelzine and pargyline as specific inhibitors. Using this technique with frozen sections of human term placenta and rat liver, the histochemical localization of monoamine oxidase A and B and benzylamine oxidase has been demonstrated.     

The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissue

The influence of fixation and tissue preparation on the immunohistochemical localization of human fibronectin in gastrointestinal tract tissue has been examined using indirect immunoperoxidase technique. The most optimal staining result with strong intensity and well defined localization was obtained on frozen sections of unfixed material. Nearly identical results with improved morphology were obtained when staining paraffin sections of tissue fixed in 96% ethanol, 96% + 1% acetic acid and absolute acetone. All other fixatives tested, 10% neutral buffered formalin. Lillie's AAF, Bouin's fixative, Clarke's fixative, 4% formaldehyde, 4% formaldehyde + 0.5% cetylpyridiniumchloride (F-CPC), 4% formaldehyde +0.1% glutaraldehyde gave unsatisfactory results. However, proteolytic digestion with pepsin of paraffin sections prior to staining of buffered formalin and F-CPCfixed material gave results comparable with those obtained on unfixed frozen sections are regards definition of the staining whereas staining intensity was decreased in some degree. No improvement was observed when using proteolytic digestion of tissue fixed in other fixatives.