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.     

Wrinkle-Free Plastic Sections for Light Microscopy

Plastic sections 0.5 to 2 μm thick are routinely used for light microscopy. Although plastic sections have several advantages over paraffin or celloidin sections, a problem that is often encountered with plastic sections is wrinkling (Fig. 1). Wrinkling occurs during staining when sections dried on glass slides are covered with stain and heated to hasten the penetration of the stain. Mounted sections heated on glass slides, but not stained, ordinarily lack wrinkles, even when examined with phase contrast optics. Similarly, mounted sections covered with stain, but not heated, lack wrinkles; unfortunately, such sections fail to stain adequately. Unmounted sections floated on heated drops of stain also lack wrinkles (Millonig 1980). Thus, it is clear that wrinkling occurs only when mounted sections are covered with stain and heated.     

In vitro adherence of lymphocytes to unfixed and fixed high endothelial cells of lymph nodes.

Rat thoracic duct lymphocytes (TDL) bind selectively to venules lined by high endothelial cells (HEV) when overlaid onto glutaraldehyde-fixed frozen sections of lymph nodes. This report describes the characteristics of TDL binding to HEV in unfixed frozen sections and compares this reactivity with that observed after fixing sections with different reagents. We found that TDL bound to unfixed HEV and that the pattern of adherence to such sections was identical to that observed when using glutaraldehyde-fixed tissue. Fixation of the sections with glutaraldehyde, however, enhanced the binding reaction. This effect was also observed when sections were treated with the diimidoester, dimethylsuberimidate (DMS) but not when methanol or formaldehyde was used. Since glutaraldehyde and DMS are each bifunctional cross-linking reagents, the results suggest that in vitro HEV adherence was facilitated under conditions in which the endothelial binding sites were present in an aggregated form.     

BIASES IN DOLPHIN AGE STRUCTURE DUE TO AGE ESTIMATION TECHNIQUE1

The use of different tooth-preparation techniques resulted in widely different estimates of age in a sample of bottlenose dolphins, Tursiops truncatus. Teeth from 30 animals were prepared using the two most prevalent techniques reported in the literature for this species, unstained sections and decalcified and stained thin sections, and the resulting paired counts of growth layers were compared. Estimates from the two methods were identical or at least placed the specimen in the same age class in only five cases, ranging in age from 2 to 22 yr. Otherwise, the results fell into one of two categories: when the estimates were close (± 3-yr difference, n= 15), counts from unstained sections generally were higher (13 cases, age from unstained sections 2-20 yr); when the counts were more disparate, estimates from stained sections always were higher (6-31 yr difference, n= 10, age from unstained sections 12-27 yr and corresponding ages from stained sections of 27-47). Previous studies of age estimation in known-age bottlenose dolphins indicate that stained sections allow accurate estimates of age and demonstrate that maximum lifespan approaches or exceeds 50 yr. In contrast, the results herein suggest that using unstained sections for age estimation may result in imprecise or biased age-structure data.     

Ancymidol-enhanced hyperhydric malformation in relation to gibberellin and oxidative stress in liquid-cultured <Emphasis Type="Italic">Narcissus</Emphasis> leaves

Summary The growth retardant ancymidol inhibited gibberellin biosynthesis and enhanced hyperhydric malformation of Narcissus leaf sections cultured in liquid medium. Superoxide dismutase activities were examined by spectrophotometry and native polyacrylamide gel analysis, and gibberellin and hydrogen peroxide levels were determined spectrophotometrically in either hyperhydric or non-hyperhydric leaf sections. In ancymidol-treated hyperhydric leaf sections, superoxide dismutase activity and hydrogen peroxide levels were higher during the initial culture period, when hyperhydric malformation occurred, than in control untreated leaf sections. At a later stage, when the meristematic centers started to form on ancymidol-enhanced hyperhydric leaf sections, superoxide dismutase activity, hydrogen peroxide, and gibberellin levels were significantly lower in hyperhydric leaf sections than in non-treated leaf sections. The changes in superoxide dismutase activities, hydrogen peroxide, and gibberellin levels appeared to be related to hyperhydric malformation and meristematic center initiation.     

A comparative study of morphometric measurements of nucleoli in uveal melanomas from electron micrographs and plastic-embedded and paraffin-embedded sections

Morphometric measurements of nucleoli were done on uveal melanomas from surviving and nonsurviving patients. The melanomas were embedded in paraffin and plastic, and measurement data from Papanicolaou-stained paraffin-embedded sections, toluidine blue-stained plastic-embedded sections and scanning transmission electron micrographs (STEM) of plastic-embedded sections were compared. The results showed that one parameter, the coefficient of variation (CV) of nucleolar area, correctly classified 80% of the cases as to survival when plastic-embedded material was used and 70% of the cases when paraffin-embedded material or STEM micrographs were used. The inverse standard deviation of the nucleolar area was a better predictor of outcome than was the CV of nucleolar area only in the paraffin-embedded sections. The nucleolar measurements were most easily and rapidly performed in the plastic-embedded sections.     

Heterogeneous staining of rat hepatocyte nuclei by HBFP technique

Synopsis The non-enzymatic histochemical technique Haematoxylin-Basic Fuchsinpicric acid (HBFP) was studied in fresh-frozen and Carnoy-fixed, paraffin-embedded rat liver sections. The hepatocyte nuclei fell into two populations and showed either a crimson red or purple staining in frozen as well as paraffin sections. The heterogeneous staining of the rat heptocyte nuclei was also present when the tissue sections were stained by Methyl Green-Pyronin stain. The differing nuclear staining was present in the isolated nuclei also. The HBFP technique, therefore, appears potentially useful when applied to liver and other tissues as well.     

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

A Method to Stain Decalcified Bone without Loss of Structural Detail

A modification of Bodian's protargol-S technique is done on 7 μm sections of decalcified bone. Light microscopic results are greatly improved when compared to either ground bone sections or decalcified bone stained routinely with hematoxylin and eosin.     

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

The application of lipid-soluble stains in plastic-embedded sections

The present study was designed to develop a routine method for direct demonstration and precise localization of lipid substances in tissue sections. A panel of lipid-rich tissues was fixed in 4% buffered formaldehyde, infiltrated, and embedded in the water-soluble plastics Technovit 7100, EFL-67, and JB-4. The use of alcohol containing fluids was avoided. Staining with the lipid-soluble dyes Sudan Black B and Oil Red O revealed excellent preservation of tissue lipids in Technovit 7100 embedded sections when compared with cryostat sections of the same tissue specimens. Lipid preservation in EFL-67 and JB-4 embedded sections was inconsistent, even when infiltration and polymerization procedures were performed at 4 degrees C. Combination of lipid-soluble dyes with the periodic acid Schiff, Jones' methenamine silver, or Gomori' reticulin method allowed for an exact localization of lipids in high-quality Technovit 7100 embedded sections. The procedure herein is easily applicable in routine histopathology practice.     

Refinement of an enzyme-linked immunosorbent assay for detecting sapstain fungi in wood

Antigens from Ophiostoma sp. C28 could be removed effectively from solid wood by grinding thin sections of wood or sawdust in buffer. Fungal antigens were more soluble when detergents (Tween 20 or Triton X100) were added to the extracting buffer. The detergent had to be subseguently removed or diluted out, because it interfered and prevented the binding of the antigen onto the microtitration plate. Good results were also obtained when the ELISA was performed directly on thin sections of wood. This latter procedure was significantly less time consuming.     

A Teflon Disk for Processing Loose Serial Celloidin Sections

Celloidin sections are routinely used for Nissl, Golgi, or Golgi-Cox staining (e.g., Glaser and Van der Loos 1981) when sections thicker than 30 μm are required. In spite of the advantages of the celloidin method (see Voogd and Feirabend 1981, Buschke 1979), processing free-floating serial sections of celloidin embedded material, which may often be preferred, is not very convenient.     

The diamond knife "semi": a substitute for glass or conventional diamond knives in the ultramicrotomy of thin and semi-thin sections

The diamond knife "semi" for ultramicrotomes was originally designed by its manufacturer (DIATOME S.A.), for cutting semi-thin sections from 0.2 micron to 2.0 micron. Cutting tests of Epon-embedded material (nervous system, myelin sheat) with this knife have shown that the quality of semi-thin sections is equivalent or better than that obtained with a glass knife, and much time could be saved during the microtomy of serial sections. The quality of thin sections (0.07 micron to 0.12 micron) is excellent and comparable to that obtained with a conventional diamond knife. Furthermore, when adjacent sections are cut thin and semi-thin (for immunochemistry or high voltage microscopy), both are excellent in that they are of uniform thickness. In conclusion, this tool has advantages for both light microscopy and transmission electron microscopy.     

An economical minichamber for immunohistochemical incubation

A simple and economical "slide-minichamber" method for incubating tissue sections with antisera in immunohistochemical (peroxidase-antiperoxidase) staining procedures is described. The technique requires only materials routinely used in the laboratory. The method permits prolonged incubation of tissue sections with antiserum at 4 degrees C or at room temperature, use of small quantities of antiserum, and simultaneous incubation of two tissue sections with the same small quantity of antiserum, thereby allowing use of very dilute antisera and conservation of antisera when availability is limited.     

Characterization of hyaluronic acid on tissue sections with hyaluronectin

An affinity immunological procedure for hyaluronic acid detection on tissue sections is described. This new, sensitive, and specific technique is based on the high affinity of hyaluronectin for hyaluronic acid, utilizing anti-hyaluronectin-hyaluronectin immune complexes. Elimination of binding when the reagent was supplemented with hyaluronic acid or when Streptomyces hyaluronidase-digested tissue sections were used emphasizes the specificity of the assay. This technique made possible accurate HA localization in embryonic mesenchyme, in neural tissue, in kidney medulla, and in tumors.     

Penetration of Stain in Ultrathin Sections of Tobacco Mosaic Virus

Purified preparations of tobacco mosaic virus (TMV) and pieces of tomato leaves infected with TMV were embedded in methacrylate or epoxy resin, sectioned, and stained with 1.0% strontium permanganate for electron microscopy. In sections containing purified and intracellular virus, the apparent length of stained particles varied directly with section thickness, indicating stain penetration beyond the surface of the section. Penetration was demonstrated also by stereoscopy. Penetration was less complete when sections were allowed to dry before staining. In most instances the number of identifiable particles per unit area was independent of section thickness but increased when both surfaces of the sections were stained instead of only one surface. Staining was prevented by thin films of methacrylate or epoxy resin placed between the virus section and staining solution. Most results supported the view that electron scattering capacity was enhanced only in particles which intersected the surface of the section exposed to permanganate.     

Staining of depolymerised DNA in mammalian tissues with methyl violet 6B and crystal violet

The paper contains an account of DNA staining with basic dyes; methyl violet 6B and crystal violet in mammalian tissue sections after RNA extraction with cold concentrated phosphoric acid. The study shows that the best staining is obtained at pHs 2.5 and 3.5. Dehydration of stained nuclei is perfect when a mixture of absolute ethanol and n-butanol is used followed by treatment of sections in isoamyl or amyl alcohol. The in situ absorption data of nuclei stained with aqueous solution of methyl violet 6B as well as with crystal violet are also presented. Possible mechanism of staining as well as an explanation for dye-leaching when sections are dehydrated through ethanol are discussed.     

The reaction of osteoclasts when releasing osteocytes from osteocytic lacunae in the bone during bone modeling

Osteocytes are released from the osteocytic lacunae when osteoclasts resorb the bone matrix during bone modeling and remodeling. It remains unknown how osteoclasts react when releasing osteocytes during bone modeling, and the fate of these released osteocytes is also unclear. Femoral mid-shafts of 2-day-old kittens were sectioned into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and examined by light microscopy (LM) and transmission electron microscopy (TEM). The sections showed many osteoclasts at the endosteum but there were no osteoblasts. There were many half-released, fully released, half-exposed, and fully exposed osteocytes on the bone surfaces. Many cell-like structures were seen in the cell bodies of osteoclasts by LM, and some semithin sections were re-sectioned into ultrathin sections for re-observation by TEM. By TEM, these were determinated to be mononuclear cells. The serial ultrathin sections showed that the mononuclear cells appeared to be engulfed in osteoclasts on one section but that the cell was connected with the bone surface of the osteocytic lacuna on another section. These results show that the mononuclear cells in the osteoclasts were osteocytes. The present study suggests that osteoclasts engulf some osteocytes but do not engulf others when releasing osteocytes during bone modeling.