Use of a Silicone Rubber (Clear Seal) as a Section Adhesive Resistant to Acid, Alkali and Heat

An optically clear silicone rubber adhesive is recommended for use in histochemical procedures in which detachment of tissue sections is likely. Procedure: Cut paraffin sections and float on a 45-50 C water bath; leave frozen sections on the microtome knife in the cryostat; spread the silicone rubber thinly and evenly over 2/3 of the slide (Clear Seal—General Electric, was used); pick up paraffin sections directly from the floatation water and frozen sections from the microtome knife with a warm slide; dry for 1.5 hr at 25 C; place paraffin sections in a 60 C oven for 0.5 hr, deparaffinize through xylene and hydrate through alcohols to water. Stain sections as desired, but avoid clearing agents before mounting after strong acid or alkaline treatment, and mount rapidly if a synthetic resin is used because of the solvent effect on the silicone rubber. Of the adhesives tried, silicone rubber is the only one capable of withstanding boiling 10% HCl for any period of time without detachment of sections.     

Floating-Out Technics for Rapid Placement of Ribbons of Serial Sections on Slides

The floating-out technic, popular for single paraffin sections, can be applied successfully to ribbons of serials by either of two procedures. (1) If spreading time for the sections is uncritical suitable lengths of ribbon for attachment to a slide are laid on water at a temperature about 8° C below the melting point of the paraffin and manipulated with a rubber bulb pipette to form a unit. This ensemble can then be picked up by the slide in much the same manner as a single section. (2) If spreading time is critical, as for objects that have had guide limes embedded with them, several ribbons are arranged on a cold, dry slide and transferred to the water as a unit. Placing the ribbons on the cold slide so that they slightly overhang one end and the sides of the slide allows them to make proper contact with the water as the slide is immersed. To facilitate controllable spreading in both methods, the water should have added to it 0.5 ml of albumen-glycerol adhesive per 100 ml. Adding water to the slide after the sections have been picked up or manipulation of the ribbons is generally unnecessary if the ribbons have been aligned accurately on the floating-out bath.     

Growth inhibition due to aged silicone rubber tubing

Growth of the obligately methylotrophic Bacterium B6/2 in continuous culture was inhibited when the mineral salts-methylamine medium was supplied through silicone rubber tubing which had been autoclaved more than 50 times. The growth inhibition was removed when the medium was supplied through new silicone rubber tubing. Growth in small-scale batch cultures was delayed or inhibited after the medium passed through the former tubing but not when it had passed through the latter. It is recommended that silicone rubber medium supply lines to chemostats are replaced periodically.     

Application of cryo-compatible antibodies to human placenta paraffin sections

The hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) -fixation and paraffin embedding technique has been described to expand possibilities for immuno-labellings due to low denaturation of proteins. In this study, the issue was addressed as to whether the HOPE technique could be a useful tool in placenta tissue-based studies when only cryo-compatible antibodies are available. Such antibodies can be used on cryostat sections only, giving results of considerably inferior morphological detail as compared to routinely fixed paraffin embedded tissue sections. Commercially available, only cryo-compatible, monoclonal antibodies against a conformational epitope of HLA-G (clone MEM-G/9) and leukocyte differentiation antigens CD56, CD163 and CD34 III were selected and applied to frozen sections, routinely formalin-fixed and HOPE-fixed paraffin sections. All tested antibodies immunolocalized their antigen on cryo sections and on HOPE-fixed but not formalin-fixed paraffin sections. The HOPE technique provides an excellent preservation of protein antigenicity together with well presented morphological details in paraffin embedded placenta tissues. The detection of native or conformation-dependent epitopes in paraffin sections expands the immunolocalization possibilities in placenta research and reproductive immunology.     

N-butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18-24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

N-Butyl methacrylate and paraffin as an embedding medium for light microscopy.

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18--24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

Handling Small Numbers of Marine Eggs Through Dehydration and Embedding

A method is described whereby small numbers of eggs can be embedded compactly. Handling through reagents is achieved in 6 mm. glass tubing having a linen mesh affixed to one end, up to and including paraffin. The egg mass is then obtained and reembed-ded in a pre-cut paraffin block in which a well has been drilled. Perfect seal of the two masses is achieved with a hot needle. Some advantages of the method are described.     

Loss of lipid to plastic tubing

14C-labeled oleic acid and (3)H-labeled monoether in a bile salt solution were perfused through three types of plastic tubing. Large proportions of lipid were lost to the walls of silicone rubber and polyvinyl chloride tubes. The major portion of the lipid lost was recoverable only when chloroform-methanol was perfused through the tubings. On the other hand, very little lipid was lost to the wall of polyethylene tubing. Polyethylene tubing should therefore be used in perfusion studies involving lipid-soluble compounds.     

Colonization of components of a model hot water system by Legionella pneumophila

A model hot water distribution network was seeded with a virulent strain of Legionella pneumophila serotype 1. Ten weeks after inoculation, components of the system, which include aluminium discs, copper, stainless steel, silicone tubing, rubber and glass beads, were examined for colonization by L. pneumophila. The samples were stained with fluorescein-labelled antibodies to the strain and were examined with scanning electron microscopy. Colonization, which was accompanied by copious quantities of a slime-like debris, was heaviest on the rubber and least on the copper. Adherence to silicone tubing and stainless steel was observed.     

Colonization of components of a model hot water system by Legionella pneumophila

A model hot water distribution network was seeded with a virulent strain of Legionella pneumophila serotype 1. Ten weeks after inoculation, components of the system, which include aluminium discs, copper, stainless steel, silicone tubing, rubber and glass beads, were examined for colonization by L. pneumophila. The samples were stained with fluorescein-labelled antibodies to the strain and were examined with scanning electron microscopy. Colonization, which was accompanied by copious quantities of a slime-like debris, was heaviest on the rubber and least on the copper. Adherence to silicone tubing and stainless steel was observed.     

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 Simple Method for Collecting and Mounting Ribboned Serial Sections of Epoxy Embedded Specimens

A simple and rapid method of handling ribboned serial sections of epoxy embedded specimens is described. Ribbons are cut from a block having the leading and trailing sides coated with contact cement. A scoop made from polyethylene tubing is used to remove a ribbon of sections from the boat of a glass or diamond knife and to transfer it to a pool of water on a microscope slide. Many ribbons (comprising hundreds of sections) can be mounted on a single slide. This method requires the construction of only one simple, inexpensive tool, the polyethylene scoop, and otherwise utilizes only items commonly available in the laboratory.     

Replacing xylene with n-heptane for paraffin embedding

Abstract In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.     

A simple method for collecting and mounting ribboned serial sections of epoxy embedded specimens

A simple and rapid method of handling ribboned serial sections of epoxy embedded specimens is described. Ribbons are cut from a block having the leading and trailing sides coated with contact cement. A scoop made from polyethylene tubing is used to remove a ribbon of sections from the boat of a glass or diamond knife and to transfer it to a pool of water on a microscope slide. Many ribbons (comprising hundreds of sections) can be mounted on a single slide. This method requires the construction of only one simple, inexpensive tool, the polyethylene scoop, and otherwise utilizes only items commonly available in the laboratory.     

Identification of oestrogen receptors in cells of paraffin-processed breast cancers by IGSS

Oestrogen receptor (ER) analysis of breast cancers by the standard dextran coated charcoal (DCC) method and the oestrogen receptor immunocytochemical assay (ERICA), shows that ERICA is more sensitive. We find that the immunogold-silver staining technique (IGSS), which is used on paraffin sections, is applicable to the ERICA antibody and that the DCC and IGSS methods have comparable sensitivity. Reasons for wishing to develop an improved method for oestrogen receptor localisation in paraffin sections and its advantages are given.     

Detection of Mycobacterium avium subsp. paratuberculosis in formalin- fixed,paraffin embedded tissues of goats by IS900 polymerase chain reaction

The objective of the present study was to optimise and evaluate a procedure for detection of Mycobacterium avium subsp. paratuberculosis in archived formalin-fixed, paraffin embedded tissue sections from cases of naturally occurring paratuberculosis in goats. A pilot study assessed 3 procedures for extraction of DNA for detection by PCR. The procedure that gave the most consistent results involved removal of paraffin by treatment with xylene and ethanol, disruption of tissue pellets by beating with zirconium/silica beads, extraction of DNA using a DNeasy kit (Qiagen) with overnight proteinase K digestion and final ethanol precipitation. This procedure was used to analyse 82 paraffin embedded tissues (44 small intestine, 38 mesenteric lymph node) with various grades of histological lesions of paratuberculosis and acid-fast bacilli (AFB) loads. The overall sensitivity of the PCR was about 72% of all samples including both paucibacillary and multibacillary lesions. The sensitivity of the assay was 87.5% (42/48) in all paraffin sections having clearly and easily demonstrable AFB. Fifty percent of the tissue sections with rarely detectable AFB were positive by PCR. There was no significant difference (<0.05) between the sensitivity of the PCR analyses carried out on intestinal and mesenteric lymph node tissues. The results of this study suggest that IS900 PCR on formalin-fixed, paraffin embedded histological sections is a practical and important tool for confirming diagnosis of paratuberculosis in goats, where fresh tissues for bacterial culture or PCR are not available due to problem of maintaining a cold chain system.     

Investigation of Tissue Preparation Conditions for Non-radioactive In Situ Hybridization: Localization of Transforming Growth Factor-α Message in Rat Kidney

A non-radioactive method of in situ hybridization was used to localize transforming growth factor- mRNA in epithelial cells of collecting ducts and tubules in rat kidney tissue sections. The intensity and specificity of staining were assessed under a variety of tissue preparation conditions, including a direct comparison of paraffin against frozen sections. Under optimal conditions, both the signal strength and the cellular localization of the growth factor message were superior in paraffin sections. The staining method could also be used to localize the message in lung tissue, indicating that the procedure is generally applicable to other tissues. Our results indicate that the use of paraffin sections for non-radioactive in situ hybridization affords a number of advantages for the localization of specific messages in tissue sections.     

Enzyme histochemical discrimination between tryptase and chymase in mast cells of human gut

We tested four synthetic substances for their histochemical value to demonstrate the catalytic activities of chymase or tryptase in mast cells in sections of human gut. Both Suc-Ala-Ala-Phe-4 methoxy-2-naphthylamide (MNA) and N-acetyl-L-methionine-alpha-naphthyl ester (alpha-N-O-Met) reacted with chymase but not tryptase in mast cells. Conversely, D-Val-Leu-Arg-MNA and Z-Ala-Ala-Lys-MNA were hydrolyzed by mast cell tryptase but not chymase. These results were confirmed by use of two inhibitors of chymotrypsin-like activity, chymostatin and Z-Gly-Leu-Phe-chloromethyl ketone (CK) and two inhibitors of trypsin-like activity, Tos-Lys-CK and D-Val-Leu-Arg-CK. Excellent staining reactions were obtained on cryostat sections of unfixed or aldehyde-fixed tissues and on paraffin sections of Carnoy-fixed tissues. For chymase, however, Suc-Ala-Ala-Phe-MNA is preferred on cryostat sections because it is more specific. On paraffin sections alpha-N-O-Met is preferred because other cells are not then stained. For tryptase, Z-Ala-Ala-Lys-MNA was more selective and more specific and is the preferred general purpose substrate on cryostat sections of aldehyde-fixed tissues and for paraffin sections. D-Val-Leu-Arg-MNA is the preferred substrate for cryostat sections of unfixed tissue. Only a limited number of mast cells showed a reaction for chymase, and these occurred mainly in the submucosa. All mast cells, however, gave a reaction for tryptase, and we recommend the use of either substrate for this enzyme for routine detection of mast cells in human tissues. Double staining for the two main mast cell proteases is most conveniently undertaken on paraffin sections of Carnoy-fixed tissues using MNA substrates for tryptase and alpha-N-O-Met for chymase.     

Comparison of two histological techniques for age determination in small cetaceans

Age estimation in odontocetes is based on counts of growth layer groups (GLGs) deposited in recording structures such as teeth. Generally, tooth sections are obtained using a cryostat microtome. However, some researchers prefer obtaining thin sections using a traditional paraffin microtome. Little information is available on the application of this technique to dolphin teeth. Our main aim was to investigate if the paraffin technique can be a viable alternative. We considered whether estimated age would be affected by preparation technique, staining method, and section thickness, while controlling for effects of species, body length, and sex. We also analyzed whether the staining method would affect readability of GLGs and age reading variability. Teeth from 86 individuals (representing seven species) were used, but not all were prepared using both techniques because sufficient teeth were not available in all cases. Although the staining method had significant effects on the estimated age using both techniques, the variability of GLG counts was small and appeared to be similar for both techniques. Using Mayer's hematoxylin stained sections of 8 μm thickness, good agreement of ages was obtained from both techniques, with more preparations classified as "good quality" for the paraffin technique. Mayer's hematoxylin provided the best contrast of the GLGs when using the paraffin technique. We conclude that the paraffin technique is viable and represents a cost-effective alternative to a cryostat microtome when preparing cetacean teeth for age determination.     

Postadhesive Technique for Archival Paraffin Sections

We present a postadhesive protocol for adhering paraffin sections of archival material to microscope slides. Appropriately posttreated sections, subsequently processed for immunohistochemistry, remained attached to the slides and were well preserved with no signs of artifacts, such as scratching and shrinkage. The immunohistochemical staining was intense and antigen-specific without nonspecific background. Specific staining intensity was equal to that produced in untreated control sections; however, the latter became partially or fully detached from the slides. The postadhesion protocol may be used with modern techniques and is recommended for reclaiming use of otherwise unsuitable paraffin sections of archival material.