Accurate Placement of Ultrathin Sections on Grids; Control by Sol-Gel Phases of a Gelatin Flotation Fluid

A 3% solution of gelatin in a petri dish at 25-30 C. provides a liquid, viscous surface upon which ultrathin sections can be floated. On cooling, the gelled substratum immobilizes the sections, allowing grids to be placed on them with any desired grid-to-section orientation. When the gelatin is remelted, the sections remain attached to the grids. After draining, traces of gelatin adhering to the grids are removed by flotation (section side down) for 30 min on 2% acetic acid at 60 C. This is followed by flotation for 3-5 min on Tris buffer, pH 7.1, and then on distilled water for 30 min—both treatments at 60 C. The technique is particularly useful for mounting serial sections.     

Movement of Pulses of Labeled Auxin in Corn Coleoptiles

The transit of indole-3-acetic acid through 20-mm sections of corn coleoptiles can be separated from processes involved in the uptake of auxin by the section and the exit of auxin from the section. Aerobic sections are supplied with an exogenous source of (14)C IAA for a limited time, and after the source is removed, a pulse of (14)C IAA moves down at 12 to 15 mm/hour. After transfer to nitrogen, movement of the pulse at the aerobic rate persists for about 10 minutes; thereafter drops to only 1 to 2 mm/hour and remains at this level during the next 4 hours. Within 2 hours, 70% of the total (14)C in aerobic sections has moved 10 mm or more down the section from the position of the initial peak, whereas after the same time in nitrogen less than 10% of the total (14)C has moved as far.During the migration down the coleoptile, the peak of radioactivity becomes broader and less distinct. This dispersion is more rapid in aerobic than anaerobic sections, but appears to be nonpolar and to occur along the existing concentration gradients. Diffusion probably contributes to this dispersion.In both inhibited and uninhibited sections, the movement of the peak, in contrast to its dispersion, is A) polar (downward) and B) independent of existing concentration gradients. Thus transit within the section possesses the fundamental properties of the overall transport system. The reduced amount of transport in inhibited sections is more likely maintained by glycolysis than by a low level of aerobic respiration dependent on the residual oxygen in the tissue.     

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.     

Quantitative histology of the rat thyroid. Influence of histologic techniques on the morphometric data

The influence of various histologic techniques on the results obtained by morphometric analysis of the rat thyroid gland was studied. The limits of thyroid follicles were more clearly defined in both silver-impregnated paraffin-embedded sections and resin-embedded semithin sections than in routinely stained paraffin-embedded sections, thus enabling more accurate measurements of thyroid structures. Due to its simplicity, the silver impregnation method is clearly useful for histomorphometric studies when large numbers of measurements are involved. C cells were easily identified in paraffin-embedded sections by immunohistochemical staining. The measurement of interstitial tissue in sections without immunostaining of C cells led to an overestimation of the volume fraction of interstitial tissue.     

Polystyrene embedding: a new method for light and electron microscopy.

Polystyrene embedments of histological specimens can be obtained with a solution of 1:14 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue, which are then glued to a Plexiglas support. Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy sections on a slide heated on a hot plate to 80 C; these can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fixed for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and given acceptable results.     

A Rapid Plastic Embedding Technique for Preparation of Three-Micron Thick Sections of Decalcified Hard Tissue

A 24 hour start-to-finish method is described for the preparation of three-micronthick sections of decalcified hard tissues. Following acetone dehydration, the tissue to be embedded is infiltrated under vacuum with a series of graded clearing solutions which approach the content of the final methyl methacrylate mixture. After overnight in a 35 C oven, the plastic is polymerizd by four hours heating at 42 C. Three-micron-thick sections are then easily prepared using a Jung microtome for high resolution histologic or detailed autoradiographic procedures.     

A method for preparing whole-body sections suitable for autoradiographic, histological and histochemical studies

A method for the preparation of whole-body sections suitable for autoradiographic and histochemical study is described. Radioactive calcium chloride or [14C]proline was injected into the abdominal cavity of a rat. Thirty-five minutes after injection of calcium chloride or 40 min after injection of proline the rat was frozen in a mixture of hexane and solid carbon dioxide and blocked in 5% sodium carboxymethyl cellulose. The carboxymethyl cellulose block was trimmed and a piece of copy paper was attached to the surface of the block with cellulose tape. Cryotome sections cut from the block were transferred from the paper to a glass slide coated with synthetic rubber adhesive. For whole-body autoradiography, sections were freeze-dried for 2 days and then placed against X-ray film. For light microscopic autoradiography, the freeze-dried sections were covered with a dried film of photographic emulsion. For histochemical use, the sections were fixed by raising the temperature to 4 C after immersion in 100% ethanol below -10 C. For histological observation, sections were postfixed with 2.5% glutaraldehyde and stained. Whole-body and light microscopic autoradiographs showed that sections so prepared could be used for the demonstration of soluble substances in whole-body sections and for detailed autoradiography at the light microscopic level, and the stained sections could be used for histological and histochemical studies.     

Conditions for the immunohistochemical demonstration of complement factor C3 in formaldehyde-fixed and paraffin-embedded renal tissues

Paraffin sections of formaldehyde-fixed renal biopsies were labeled for complement C3 by a polyclonal rabbit antibody to human complement C3, by the peroxidase-antiperoxidase complex (PAP) and the avidin-biotin peroxidase complex (ABC) techniques, respectively. All tissues had C3 deposits according to direct immunofluorescence on fresh frozen sections. Staining for muramidase was introduced as an intrinsic control for the degree of tissue proteolysis after the necessary trypsin digestion prior to the immunoenzyme labeling. The results indicated that even minute deposits of C3 could be detected in paraffin sections by the ABC method, which was more sensitive than the PAP technique; the ABC method allowed a maximal dilution of 1:2,400 of the primary antibody as compared to 1:800 for the PAP technique.     

Polystyrene Embedding: A New Method for Light and Electron Microscopy

Polystyrene embedments of histological specimens can be Obtained with a solution 1 : 4 polystyrene-toluene, 5% benzyl alcohol and 1% dibutyl phthalate, allowing the solvent to evaporate in polyethylene containers for 2-3 days at 58 C. The resulting blocks are easily cut into truncated pyramids, each containing a piece of tissue. which are then glued to a Plexiglas support Drying is completed at 80 C for 20 hr. The pyramids can then be sectioned to produce thick sections, with a steel knife or to produce semi- or ultrathin sections with a glass knife. A 10% paraldehyde solution is used to mount the light microscopy dons on a slide heated on a hot plate to 80 C; those can be treated with the same techniques used with paraffin sections. The results are of high quality. Semithin sections of tissues fired for electron microscopy can be stained directly after mounting, or by a wider range of stains once the polystyrene has been removed by organic solvents. In electron-microscopy, the ultrathin sections obtained with the usual techniques are highly electron beam-resistant and give acceptable results.     

Synthesis and turnover of nitrate reductase in corn roots

The induction and reinduction of nitrate reductase in root tip or mature root sections show essentially a similar pattern: a lag, a period of rapid increase in enzyme activity and finally a period of relatively minor change. Both inductions are sensitive to 6-methylpurine and cycloheximide. Kinetic studies with 6-methylpurine suggest that the half-life of the messenger RNA for nitrate reductase in both sections is about 20 minutes. The rate of decay of nitrate reductase activity induced by transfer to a nitrate-free medium is slower in root tips (t½ = 3 hours) than in mature root sections (t½ = 2 hours). The enzyme from mature root sections is also less stable to mild heat treatments (27 C; 40 C) than the enzyme from root tip sections. The results indicate that factors regulating enzyme turnover show important changes as root cells mature and may be significant in determining steady state levels of the enzyme.     

Superficial Warming of Epoxy Blocks for Cutting of 25-150 μm Sections to be Resectioned in the 40-90 nm Range

An improved method is described in which tissue areas can be initially identified in thick sections by light microscopy and isolated for subsequent ultrathin sections and observation by electron microscopy. This is achieved by embedding in hard Epon which can be sectioned at 25-150 μm on a sliding microtome after softening the blockface by applying a 60-70 C tacking iron to its surface immediately before each section is taken. The thick sections are then mounted on plastic slides to enable light microscopic selection of areas to be observed by electron microscopy. The selected areas are remounted on faced Epon blanks and resectioned at less than 50 nm. This technique makes it possible to obtain thick sections while maintaining an Epon hard enough for good serial ultrathin sections.     

Effects of time and temperature during attachment of sections to microscope slides on immunohistochemical detection of antigens.

To study the effects of time and temperature on attachment of tissue sections to microscope slides, we examined the intensity of immunohistochemical staining of selected antigens in nine different neoplastic and normal tissues after attaching sections at different times and temperatures. Typically, both the temperature and time are minimized when tissue sections attached to slides; however, suboptimal times and temperatures during attachment may result in either loss of tissue due to poor attachment or the necessity for inconvenient staining regimens. Using standard immunohistochemical techniques, 5 microm tissue sections were attached at 58 degrees C for 1, 4 and 24 hr. In a separate study, 5 microm tissue sections were attached for 16 hr at 58, 68 and 80 degrees C. The intensity of staining decreased slightly when the tissue sections were heated at 80 degrees C for 16 hr, but there was little or no decrease when tissues were heated at 68 degrees C or lower for 16 hr, or at 58 degrees C for up to 24 hr.     

Staining Paraffin Sections Without Prior Removal of the Wax

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10-20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.     

Evaluation of real-time quantitative PCR for identification and quantification of Chlamydia pneumoniae by comparison with immunohistochemistry.

Chlamydia pneumoniae is a common cause of community-acquired pneumonia and it has been associated with atherosclerosis. C. pneumoniae has usually been diagnosed by serology using a microimmunofluorescence test, but more recently polymerase chain reaction (PCR) has been viewed as an advantageous alternative. We developed a quantitative real-time PCR for detection of C. pneumoniae. Primers were targeted for the pmp4 gene, and the PCR fragment was detected real-time with a fluorescence resonance energy transfer probe set using a LightCycler instrument. The PCR was used on DNA released from 50 microm sections of paraffin-embedded formalin-fixed lung tissue from experimentally infected mice. Thereby, the number of C. pneumoniae genomes was determined. To our knowledge this is the first time quantification of C. pneumoniae DNA has been attempted on paraffin-embedded formalin-fixed tissue. C. pneumoniae-specific immunohistochemistry (IHC) was done on 5 microm sections adjacent to the sections used in PCR, and the number of inclusions were counted in each section. Good correlation was found when comparing results from PCR and IHC, which is in contrast to many previous studies.     

Isolation of Avian Trypanosomes by Selective Centrifugation and Subsequent Processing for Electron Microscopy

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10–20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.     

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.     

Demonstration of dehydrogenase activities in paraffin sections.

The possibility of demonstrating the activity of respiratory enzymes in paraffin sections was studied. Unfixed pieces of nervous tissue were incubated at 4 degrees C, 20 degrees C, 37 degrees C and 56 degrees C for various periods ranging from 1 to 24 hours. After dehydration, the tissue pieces were mounted in paraffin. The paraffin sections obtained there of were then tested with respect to the range of penetration of the substrate into the incubated tissue samples (as judged from the resulting histoenzymic reaction), and for the distinctness with which the localization of the histochemic reaction could be assessed. From the results it may be concluded that it is possible, under well defined conditions, to demonstrate the activity of dehydrogenases in paraffin sections. The resulting morphological pictures permit a much better localization of the histoenzymic reaction products than those obtained from cryostat sections. Optimal results are obtained when tissue fragments, about 1 mm in diameter are incubated for 24 hours at 4 degrees C.     

Evidence for tertiary structural RNA-RNA interactions within the protein S4 binding site at the 5''-end of 16S ribosomal RNA of Escherichia coli.+.

Evidence is presented for tertiary structural interaction(s) (interactions(s) between two regions of an RNA molecule that are widely separated in the RNA sequence) within the 5'-one third of the 16S ribosomal RNA of Escherichia coli that constitutes the binding site of protein S4. The two main interacting RNA regions were separated by about 120 nucleotides (sections Q to M) of the 16S RNA sequence. A second, smaller gap, of 13 nucleotides, occurred within section C". The two main interacting regions contain about 150 nucleotides (sections H" to Q) and 160 nucleotides (sections M to C"). They are folded back on one another and, especially in the presence of protein S4, are strongly protected against ribonuclease digestion. The intermediate region (sections Q to M), however, is relatively accessible to ribonucleases in the S4-RNP. By partial removal of subfragments from the RNA complex it was possible to localise the two main interacting sites within sections H" - H and sections I" - C". Three main criteria for the specificity of the RNA-RNA interactions were invoked and satisfied. The possibility of other tertiary structural RNA-RNA interactions occurring in other regions of the 16S RNA is discussed. Finally, all the structural information on the S4-RNP is summarised and a tentative model is proposed.     

A Method for Preparing Whole-Body Sections Suitable for Autoradiographic, Histological and Histochemical Studies

A method for the preparation of whole-body sections suitable for autoradiographic and histochemical study is described. Radioactive calcium chloride or [¹⁴Clproline was injected into the abdominal cavity of a rat. Thirty-five minutes after injection of calcium chloride or 40 min after injection of proline the rat was frozen in a mixture of hexane and solid carbon dioxide and blocked in 5% sodium carboxymethyl Cellulose. The carboxymethyl cellulose block was trimmed and a piece of copy paper was attached to the surface of the block with cellulose tape. Cryotome sections cut from the block were transferred from the paper to a glass slide coated with synthetic rubber adhesive. For wholebody autoradiography, sections were freeze-dried for 2 days and then placed against X-ray film. For light microscopic autoradiography, the freeze-dried sections were covered with a dried film of photographic emulsion. For histochemical use, the sections were fixed by raising the temperature to 4 C after immersion in 100% ethanol below -10 C. For histological observation, sections were postfixed with 2.5% glutaraldehyde and stained. Wholebody and light microscopic antoradiographs showed that sections so prepared could be used for the demonstration of soluble substances in wholebody sections and for detailed autoradiography at the light microscopic level, and the stained sections could be used for histological and histochemical studies.