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.     

The use of glycol methacrylate embedding for studies of chromatin distribution and fine structure in salmon spermatid nuclei.

Glycol methacrylate has been used as an embedding medium for studies of spermiogenesis in the salmon. DNA and basic proteins are shown to stain with the same specificity in thick (0.5-1.0 mu) sections of GMA-embedded salmon testes as in sections of comparably fixed, paraffin-embedded testes. Stain can be localized far more precisely in GMA sections than in paraffin sections due to the thinness of the sections and to the excellent structural preservation of nuclei. In addition, ultra-thin sections of GMA-embedded salmon testes can be observed with the electron microscope, and this permits exact correlation between nuclear fine structure and chemical composition in consecutive sections of the same nuclei.     

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.     

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 sections. The technique is compatible with histochemical staining of enzymes.     

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.     

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.     

Application of glial fibrillary acidic protein immunohistochemistry in the quantification of astrocytes in the rat brain

Immunohistochemical staining for glial fibrillary acidic protein (GFAP) was employed as a tool for quantification of astrocytes in the rat brain. One-micron-methacrylate sections were prepared from 70-micron slices stained for GFAP by using a preembedding staining procedure. Numbers/unit area of astrocytes and nonastrocytes were determined for cortex, corpus callosum, and hippocampal neuropil. In each, counts from GFAP-stained, toluidine-blue-counterstained sections were compared with counts obtained from sections stained with toluidine blue alone. Numbers of nonastrocytes and total glia in all three regions were comparable in both groups of sections. Astrocyte counts in the cortex and hippocampus also showed no significant differences between the two groups. In contrast, the number of astrocytes in the corpus callosum was significantly lower in GFAP-stained, toluidine-blue-counterstained sections than in sections stained with toluidine blue alone. GFAP immunohistochemistry is a useful tool for the quantification of astrocytes in semi-thin plastic sections of rat brain.     

Interpretation of electron micrographs of single and serial sections

A method of securing serial sections for electron microscopy is described. Serial sections present certain anomalies of interpretation of a nature such that a complete and detailed three-dimensional reconstruction of the sectioned tissue cannot be made. These anomalies are discussed, as well as those which have been encountered in the interpretation of single sections. Observations of the following kinds have been made in an attempt to elucidate the interpretation of single and serial sections: differing methods of mounting adjacent sections, observation of the same section by high-angle stereoscopy, and examination of sections which have been shadowed prior to and subsequent to electron microscopy. It is found that the appearance of sections is independent of the choice of side to be placed against the formvar films. Stereoscopy shows that the appearance of fine structures is strongly dependent upon the direction of the penetrating electron beam with respect to the plane of the structures. Stereoscopy, combined with shadowing, shows quantitatively that extensive sublimation of polymer occurs upon normal exposure in the electron microscope. Observation of sections shadowed prior to electron microscopy indicates that varying amounts of material are removed between sections by the action of microtomy; i.e., it is probable that the sum of the thicknesses of several serial sections is considerably less than the total thickness of material removed from the block. It is believed that this effect, combined with the effect of sublimation, aids in explaining the failure of adjacent sections to exhibit continuity in their detailed structures.     

Application of heat-induced antigen retrieval to aldehyde-fixed fresh frozen sections.

We applied the heat-induced antigen retrieval (HIAR) to aldehyde-fixed fresh frozen sections based on a new approach (i.e., a rapid and complete immobilization of antigen followed by heating). Frozen sections were fixed with 10% formalin in 0.1 M cacodylate buffer (pH 7.4) containing 25 mM CaCl(2) for 30 min, or with 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 min at room temperature, and then autoclaved in 20 mM Tris-HCl buffer (pH 9.0) for 10 min at 120 C. Both fixatives yielded good tissue structure after autoclaving. In the sections fixed with formalin containing CaCl(2), 20 of 22 antigens located in the nucleus, cytoplasm, membranes, and extracellular matrix greatly recovered their antigenicity after autoclaving; only two antigens exhibited stronger immunoreaction in acetone-fixed fresh frozen sections than these sections. Heating also retrieved the immunoreactivity of at least 14 antigens in the sections fixed with glutaraldehyde. We used the similar procedures to localize ligand-free estrogen receptor alpha (ERalpha) and glucocorticoid receptors (GR). Mouse uterine cells exhibited almost the same nuclear ERalpha immunostaining regardless of the hormonal status in glutaraldehyde-fixed fresh frozen sections and unliganded GR was localized mainly in the nucleus of mouse hepatocytes in fresh frozen sections fixed with 20% formalin containing 50 or 75 mM CaCl(2) at 40 C, after autoclaving. These results demonstrate that HIAR is useful for the immunohistochemistry of many antigens in aldehyde-fixed fresh frozen sections.     

激光微切割与定量PCR技术分析肾脏病理切片RNA

采用激光微切割与定量PCR技术,分析使用不同提取方法从不同固定方法固定的病理切片中提取的RNA.用70%乙醇、丙酮、甲醇、4%多聚甲醛固定肾脏冰冻切片,使用激光微切割技术切取肾小球,用硫氰酸胍方法(guanidinethiocyanatemethods,GTC)和Trizol试剂方法提取RNA,使用Taqman定量PCR方法分析比较各组RNA的量;选取丙酮固定的石蜡切片,使用激光微切割技术切取肾小球,采用RNA裂解液提取RNA,使用Taqman定量PCR方法,比较石蜡切片和冰冻切片中RNA含量.结果显示:提取沉淀性固定剂如乙醇、丙酮、甲醇固定的冰冻切片的RNA时,2种提取方法和3种固定方法对RNA含量的影响都无明显差异;但在提取4%多聚甲醛固定冰冻切片时,使用Trizol提取RNA含量明显高于使用GTC方法,且其含量与沉淀性固定剂固定的切片RNA含量无明显差异.石蜡切片中经激光微切割肾小球的RNA含量与冰冻切片经激光微切割肾小球的RNA含量无明显差异.结果提示:切片的固定方法和RNA的提取方法是影响切片RNA提取量的主要原因.     

A new method of preparing embeddment-free sections for transmission electron microscopy: applications to the cytoskeletal framework and other three-dimensional networks

Diethylene glycol distearate is used as a removable embedding medium to produce embeddment -free sections for transmission electron microscopy. The easily cut sections of this material float and form ribbons in a water-filled knife trough and exhibit interference colors that aid in the selection of sections of equal thickness. The images obtained with embeddment -free sections are compared with those from the more conventional epoxy-embedded sections, and illustrate that embedding medium can obscure important biological structures, especially protein filament networks. The embeddment -free section methodology is well suited for morphological studies of cytoskeletal preparations obtained by extraction of cells with nonionic detergent in cytoskeletal stabilizing medium. The embeddment -free section also serves to bridge the very different images afforded by embedded sections and unembedded whole mounts.     

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.     

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.     

Automatic grinding of undecalcified bone sections with exact adjustment of thickness]

A new grinding machine for preparing thin undecalcified bone sections after methylmethacrylate embedding is described. About 20 rather small bone sections can be ground at the same time; bigger specimens, up to 8 cm of length, are allowed. Bone sections are mounted on a cylindrical specimen holder by an adhesive film. Then the final thickness of the sections is exactly adjusted by screwing three rubies out of the holder's bottom. Now the prepared holder is set in a guide ring on a turntable carrying a rough ended glass plate. The desired thickness of the sections is reached as soon as the three rubies touch the glass surface. The variation in the thickness of the sections is less than +/- 3 micron. The machine is simply constructed, easily to handle and rapidly to clean.     

Short Nissl Staining for Incubated Cryostat Sections of the Brain

Nissl stain often binds poorly to cryostat sections which have been incubated in solutions of radiolabeled ligands. Such incubation is used in receptor autoradiography of the brain when using the in vitro method. We have developed a rapid (16 min) modification of Nissl staining for sections that bind stain poroly, e.g., incubated sections. The method stains well sections which cannot be stained with other rapid Nissl staining methods.     

Effects of Mounting Media on Fading of Toluidine Blue and Pyronin G Staining in Epoxy Sections

Available mounting media cause fading of histological preparations over time. A study was designed to find the most suitable medium for durable mounting of Araldite embedded semithin sections of rabbit cerebral cortex stained with toluidine blue and pyronin G. Among four synthetic mounting media tested, only DePeX prevented fading of the sections during the first month. All mounting media tested helped preserve staining intensity after one month, since the fading rate after one year is only about half that in sections prepared without mounting medium. The average optical density of sections after one year was higher in preparations mounted with DePeX than in sections treated with the other mounting techniques tested in this study. After one year, the average optical density of sections mounted with DePeX had decreased approximately 20%.     

Split Nucleoli as A Source of Error in Nerve Cell Counts

The number of nerve cells in a given ganglion or nucleus is usually determined by counting the nucleoli in serial sections. The possibility that nucleoli may split and appear in more than one section is recognized as a source of error. A determination of the value of this error was made as follows; from nodose ganglia of four cats were cut serial transverse sections in which the sections varied in thickness. Thus from one ganglion, four sections were cut at 12 μ, then six at 9μ, and eight at 6μ. The process was repeated over and over until the entire ganglion was sectioned. The other ganglia were sectioned similarly. After mounting and staining, separate counts were made of the nucleoli of each given ganglion from the sections of different thicknesses. If nucleoli split according to theoretical expectations, the percentage of nucleoli split in thick sections should be less than the percentage split in thinner sections and the counts based on the sections of different thicknesses should vary accordingly. The results obtained indicate that the counts from thin sections do not differ appreciably from counts from much thicker sections, i. e., the thickness of the sections does not affect the count. It is, therefore, concluded that no correction should be made for split nucleoli if the sections are around 10 μ in thickness and none but distinct and definite nucleoli are counted.     

Fractionation of proteins of pea stem sections during root formation and its inhibition by kinetin and ethionine

Changes in the protein constituents in pea stem sections during root formation and its inhibition by kinetin and ethionine were studied. Only quantitative differences in the protein fractions separated on DEAE-cellulose column were noted. The formation of foci of meristematic cells in pericycle was accompanied by an increase in the amount of fraction “l”. This fraction disappeared rapidly in sections where root formation either did not occur (internodial sections without nodes) or was inhibited by ethionine (stem sections with basal and apical nodes). Incubation of stem sections in a kinetin solution for 16 hours after cutting of stems partially preserved fraction “l”. The increase in the amount of fraction “l” was one of the first metabolic changes in root-forming pea stem sections after cutting of stems.     

Improved correction of quantitative nuclear DNA (ploidy) measurements in tissue sections

OBJECTIVE: To evaluate, using a computer model, the advantages for ploidy measurements of selecting only center-containing sections of nuclei in ultrathin, very thick or relatively thick tissue sections. STUDY DESIGN: The computed corpuscle sectioning program was run on a personal computer. Its synthetic data were corrected by a variety of correction algorithms. RESULTS: When only center-containing sections of nuclei were selected in ultrathin sections, spherical nuclei could be corrected perfectly, and mildly prolate ellipsoidal nuclei with a selection bias in favor of elliptical nuclear section profiles could be corrected with high fidelity. Ultrathin sections most faithfully represented the true height of the peak of highest ploidy and showed better peak discrimination than other choices of section thickness, but small sample size, wavy sections, markedly inhomogeneous intranuclear DNA distribution and oblate ellipsoidal nuclei represented significant limitations of this approach. As nuclear prolation increased, peak definition worsened, and the peak of highest ploidy was falsely shortened. Results were unaffected by errors in the estimation of section thickness when an internal diploid standard was used. The effect of variable internuclear DNA concentration in mildly or moderately prolate ellipsoidal nuclei was nil. The choice of correction algorithm was unimportant, except that the reference curve method was better able to analyze oblate ellipsoidal nuclei, wavy sections and nuclei with inhomogeneous intranuclear DNA, and provided superior insight into nuclear and section parameters. Thick and very thick sections did not require correction and, unlike ultrathin sections, were immune to markedly inhomogeneous intranuclear DNA distribution, to nonspherical nuclear shape and to focal variation in section thickness (waviness), but (in relatively thick more than in very thick sections) the height of the peak of highest ploidy was falsely shortened, often markedly, and peak definition was worse. CONCLUSION: Choice of section thickness and selection of only center-containing nuclear sections for analysis with a bias in favor of elliptical nuclear section profiles in ultrathin sections are very important for optimal results; the choice of correction algorithm is less important.