Embedded Tissues Research Articles

A modification of the Humphrey’s stain for epoxy sections: A suitable alternative to toluidine blue for routine section staining

We have abandoned the use of toluidine blue as a routine stain for light microscopy of epoxy embedded tissues. As an alternative, we utilize a three-component stain described by Humphrey and Pittman (1974) with minimal modifications to render the procedure substantially more timely. Numerous publications in the early 60’s through mid 70’s proposed a variety of polychromatic stains for light microscopic evaluation of epoxy sections. Most of these reports cited the difficulties involved in using a reliable method of staining epoxy sections in a timely and non-cumbersome fashion. One of the most successful stains to address these concerns is toluidine blue, first described by Trump el al. in 1961, which is undoubtedly the stain of choice in most laboratories. We have found that the differentiation of stained tissues when using the Humphrey’s stain is superior to that rendered by toluidine blue. This stain differentiates cellular detail from connective tissue in brilliant fashion as most cells will appear in various shades of blue (quite similar to shades produced by toluidine blue) and collagen appears a vivid deep pink to bright red.

Facilitating Image Analysis of Glycolmethacrylate Embedded Tissues with Tissue- and Resin-Selective Dyes, Chosen by a Numerical Structure-Staining Relationship Model

A method facilitating recording of macroscopic images from glycolmethacrylate (GMA) embedded tissues and tissue-free sections is described. This method used dyes that selectively stain only tissue, only resin, or both, but in contrasting colors. The dyes were selected on the basis of simple numerical parameters like ionic weight, charge, and log Poct.Contrasting colors were readily converted into contrasting gray values and were so recorded for morphometric investigations, permitting areas of tissue and overall areas of resin sections to be easily measured with animage analysis system. The time saving compared to manual procedures was substantial, about 50%.We found that the small hydrophilic dyes (<550 Da) quickly stained tissues and could readily be removed from the GMA matrix by water washes. Hydrophilic dyes of intermediate size (550<IW<1000 Da) stained both the tissue and the GMA matrix, giving poor contrast between tissue and matrix. In consequence, intermediate sized dyes were suitable only for tissue-free GMA sections or in combination with small hydrophilic dyes of contrasting colors. Hydrophobic dyes proved to be unsuitable. The staining had a negligible effect on the dimensions of GMA sections. (The J Histotechnol 16:107, 1993)

Retrospective DNA analysis using fixed tissue specimens.

The recent explosion of scientific and technical knowledge in the field of molecular biology has allowed us to make important advances in our understanding of the molecular basis of many human diseases. This technology has now entered the clinical laboratory where identification of specific genetic sequences can aid in the precise diagnosis of hematologic and other malignancies, inherited diseases, specific infectious agents, and inherited predisposition to disease. In addition, it can be applied to prenatal diagnosis, paternity testing, identification of minimal residual disease following treatment, and assessment of drug sensitivity or resistance. In many cases in diagnostic pathology, the need for molecular analysis often is not realized until after a critical tissue specimen has been fixed, embedded, and examined microscopically. Thus, there is a clear need for development of techniques that would allow the retrospective study of archival tissues that have been fixed and embedded in paraffin. This review examines in depth those factors which influence the quality of the DNA available from fixed embedded tissues and discusses the usefulness of polymerase chain reaction amplification in obtaining sufficient diagnostically useful DNA from archival specimens. It is hoped that this review will aid the diagnostic pathologist interested in the application of molecular techniques in the retrospective study of fixed embedded tissues.

Identification of Infectious Agents in Epoxy Embedded Tissues

High resolution light and immunolight microscopy were carried out on a series of human tissues containing infectious agents embedded in Spurr plastic. Studies in our laboratory demonstrated that bacteria, fungi, and protozoans can beclearly delineated in plastic embedded tissues with conventional and special stains. The resolution far exceeds that attained from paraffin embedded tissue, providing the pathologist with greatly improved image information. The technique can also be applied to both fresh formalin fixed tissue and tissue extracted from paraffin blocks. (The J Histotechnol 15:293, 1992)

Laser (micro)probe mass spectrometry (LAMMS): Achievements and future trends in life sciences

Laser microprobe mass spectrometry (LAMMS) has by now become an established technique amongst others in the field of microprobe analysis with its own figures of merit and limitations. Its sensitivity for many trace elements is rather high and, in some instances, even exeeds the detection limits of SIMS (e.g. Pb). The ease and speed of operation and the possibility to incorporate classical or nonclassical (UV) transmission light microscopy for imaging tissue sections (LAMMA 500) has made it an useful technique in the microprobe analysis of e.g. embedded tissues or small particles. The number of original reports has reached nearly 1000 (about 350 related to life sciences).

Polymerization of Methacrylates: “Bubble-Hole” Artifact Reconsidered with Bone Morphometry

“Bubbles” within plastic embedded tissues have been related to entrapped oxygen or other gas produced during decomposition of the polymerization initiator. The phenomenon is maximized with bone, a tissue that requires plastic embedment for the study of its mineralization. Forty undecalcified bone biopsies with various amounts of cancellous bone from rheumatologic patients were studied morphometrically with an automatic image analyzer. The volumetric density of bone, the parameters reflecting the spatial arrangement of trabeculae, and the number of holes per section were determined. No correlation was observed between the tissue area and the number of holes. On the contrary, highly significant correlations were seen between the number of holes and the bone volume or the number of trabeculae per mm. A negative correlation was obtained with the mean distance between two trabeculae. The trabecular network creates a cellulation of the marrow space in a number of interconnected microcavities in which shrinkage produces true holes. This study points out the influence of misunderstood physical factors during plastic embedding and shows that the tissue architecture by itself may cause artifacts. (The J Histotechnol 15:51, 1992)

Double immunogold localization of opsin and actin in the cilium of developing mouse photoreceptors

A 9 + 0 cilium represents the only connection between the light-sensitive rod outer segment (ROS) and the visual cell body. Differentiation of a ROS derives from a remodeling of the plasma membrane at the distal end of the cilium. Prior to this event, an actin-rich domain can be demonstrated within the distal cilium using immunocytochemical techniques. This actin is in the filamentous form and is also observed in mature photoreceptors where it has been implicated in ROS disc morphogenesis. In separate studies, the visual pigment protein, opsin, has also been localized to the distal ciliary membrane before disc synthesis begins. For the current report, we have used double label immunoelectron microscopy to investigate the presence of opsin and actin in the cilia of developing mouse photoreceptors during the period preceding ROS differentiation. Initially, we used post-embedding immunolabeling for the localization of both proteins on ultrathin sections of Lowicryl embedded tissues. However, increased sensitivity for the detection of membrane opsin was obtained when the retinas were immersion labeled prior to resin embedment. Although it remains unclear whether the appearance of ciliary opsin and actin are synchronized, the results of this study confirm that opsin and actin are each sequestered within their respective ciliary domains prior to the differentiation of an ROS.

Use of immunogold-silver staining to visualize antibody-antigen complexes on LR white embedded tissues prior to electron microscopy

Use of immunogold-silver staining to visualize antibody-antigen complexes on LR white embedded tissues prior to electron microscopy

Use of Thick Histochemical Preparations, Whole Mount Cultured Cells or Thick Sections from Embedded Tissues, for Three-Dimensional Observation by Ultrahigh Voltage Electron Microscopy

We have observed very thick biological specimens, both whole mount cultured cells and thick sections from embedded tissues, which were stained with histochemical reactions for specific cell organelles, by means of high or ultrahigh voltage electron microscopy (UHVEM).Thick histochemical specimens, both whole mount cultured cells and semithin sections from embedded tissues were used. Two cell strains in culture, one established CHO-K1 cell line and the other primary culture cells of adult Wistar rat hepatocytes were used. Culture cells were seeded onto formval coated gold meshes and incubated in CO2 incubator. CHO-K1 cells were cultured in Ham’s F12 medium containing HRP (1 mg/ml), while rat hepatocytes were in L-15 medium containing Clofibrate (0.2 mM). The cultured cells were stained with DAB reaction, dried in a critical point dryer (Hitachi HCP-1). Thick sections (0.2-1.0 μm) from DEHP fed rat livers, fixed in 2.5% glutaraldehyde, stained with DAB reaction, postfixed in 1% osmium tetroxide and embedded in Epon, or semithin Epon sections (0.2μm) from the pancreases of 3H-thymidine or 3H-uridine injected mice, fixed doubly, radioautographed with Sakura NR-H2 emulsion.

Immunocytochemical localization of proteins utilized in the formation of outer dense fibers and fibrous sheath in rat spermatids: An electron microscope study

Affinity purified antibodies prepared against proteins isolated from fibrous sheath (FS) and outer dense fibers (ODF) were utilized in an immunocytochemical study of spermatids at various steps of spermiogenesis. This study, using the immunogold technique, was performed on sections of Epon or Lowicryl embedded tissues examined with the electron microscope. In the case of FS antibodies there was a selective immunoreactivity of the FS itself from step 10 onwards, but no reactivity over the plasma membrane associated FS anlagen. In addition there was a diffuse immunoreaction over the cytoplasmic matrix from step 9 until step 18 of spermiogenesis but no reactivity over the various types of dense bodies (e.g., granulated bodies, reticulated body, etc.) seen in the cytoplasm of these spermatids. In the case of ODF antibodies the ODF were immunolabeled throughout their development from step 11 onward. In addition to a diffuse immunoreactivity of the cytoplasmic matrix of spermatids from step 9 until step 18 of spermiogenesis, there was an immunolabeling of "granulated bodies." These bodies appeared in relation to ER cisternae during steps 10-14, increased in number and size during steps 15-17 and decreased in number thereafter leaving only a few coarsely granulated bodies in the residual cytoplasm which detached from late step 19 spermatids. No other cytoplasmic structures were labeled with the ODF antibody-gold complexes. Thus the granulated bodies appeared to serve as a transitory storage site for some proteins destined to form ODF, a major cytoskeletal element of the tail of rat spermatozoa.

An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate

Immunohistochemical localization of type I and type II collagens was examined in the rat mandibular condylar cartilage (as the secondary cartilage) and compared with that in the tibial growth plate (as the primary cartilage) using plastic embedded tissues. In the condylar cartilage, type I collagen was present not only in the extracellular matrix (ECM) of the fibrous, proliferative, and transitional cell layers, but also in the ECM of the maturative and hypertrophic cell layers. Type II collagen was present in the ECM of the maturative and hypertrophic cell layers. In the growth plate, type II collagen was present in the ECM of whole cartilaginous layers; type I collagen was not present in the cartilage but in the perichondrium and the bone matrices. These results indicate that differences exist in the components of the ECM between the primary and secondary cartilages. It is suggested that these two tissues differ in the developmental processes and/or in the reactions to their own local functional needs.

Fluorescence of Plastic Embedded Tissue Sections After Curcumin Staining

The natural dye, curcumin (C.I. 75300) from turmeric, is obtained from the roots of Curcuma longa (Lillie 1977). Curcumin has scarcely been applied for histological work, and its fluorescence seems to have been overlooked. During the course of studies on fluorescent aluminum complexes (Del Castillo et al. 1987) we realized that this dye induces a green fluorescence of chromatin (Stockert et al. 1989). In this note we describe the fluorescence reaction of curcumin on semithin sections of olastic embedded tissues.

Silver Acetate Autometallography: An Alternative Enhancement Technique for Immunogold-Silver Staining (IGSS) and Silver Amplification of Gold, Silver, Mercury and Zinc in Tissues

Silver lactate autometallography in immunogold silver staining (IGSS) usually requires development in darkness to avoid excessive background staining. Our alternative method of silver enhancement of IGSS on paraffin sections from Bouin's or formalin fixed pancreas uses silver acetate in combination with hydroquinone in low pH buffer. The modification was tested with a range of antibodies in normal and diseased tissues (all routinely fixed and paraffin embedded), in acetone postfixed cryostat sections, and in semithin sections of glutaraldehyde fixed and resin embedded tissues. Silver acetate autometallography was also tested in various systems for the visualization of tissue metals like sulfides and selenides of mercury and zinc, silver, and gold. Comparisons between sections exposed to silver lactate and the silver acetate developer showed no significant difference in the number of structures stained. The degree of background staining was often lower when silver acetate was to used as the ion donor, especially with IGSS. The advantage the technique described here is that the development process can be controlled, using normal bright field light microscopy. (The J Histotechnol 11:213, 1988.)

In-depth limitation of the Lamma 500 for the in situ localization of organic compounds in biological embedded tissue samples.

The objective of this study was exploration of the potential, offered by the laser microprobe mass spectrometer (LAMMA), for the in situ localization of organic targets in embedded tissues by means of structurally relevant ions. A series of model systems was designed to evaluate stepwise the analytical problems involved. A preliminary screening pointed to the position of the target in comparison to the actual sample surface as a determining parameter. Refined simulations were carried out with sandwich samples, consisting of an epon carrier (thickness 1 micron), coated with microcrystalline targets and covered with a layer between 5 and 50 nm of different materials (epon, carbon, formvar). In addition to the stimulated conversion of molecular into fragment ions, the presence of a barrier leads to a drastic loss of sensitivity (20-50 x) and unacceptable degradation of the mass spectrometric quality (resolution, calibration).

Histological demonstration of immunoreactive metallothionein in rat and human tissues.

Antibody to rat liver metallothionein prepared by the method of Brady and Kafka (1979) was used to localise immunoreactive metallothionein using a sensitive DNP hapten sandwich technique applied to formalin fixed wax embedded tissues. Rat tissues examined were liver, kidney and small intestine, taken from normal animals, from animals fasted after receiving either an oral dose of water, or 1 ml zinc acetate solution either orally or by intraperitoneal injection, (3-4 mg Zn++/Kg body weight). Human tissues examined were 6 histologically normal liver biopsies and small intestine including histologically normal jejunal biopsies and samples of ileum obtained at operation. Pathological tissue including liver from cases of Indian childhood cirrhosis with copper retention and ileum from cases of inflammatory bowel disease were also examined. Immunoreactive metallothionein (IMT) was found in both rat and human liver localised in the hepatocyte cytoplasm, nucleus, sinusoids and canaliculi. In some livers IMT was found in the portal and hepatic veins. In the small intestine the IMT was localised consistently in the enterocyte cytoplasm and nucleus, and in the basement membrane region. The rat kidney IMT was localised in the cytoplasm of the distal convoluted tubules the collecting tubules and the ducts of Bellini. The distribution of IMT in rat tissues showed changes associated with fasting, stress and zinc administration. In man, inflammatory bowel disease appeared to decrease the intestinal IMT and no significant difference was seen when patients had received steroid therapy. The greatest amounts of IMT were seen in the control group of patients. The distribution of IMT in human liver in Indian childhood cirrhosis did not correspond with that of copper associated protein.

An immunocytochemical study of T-cell lymphomas using monoclonal and polyclonal antibodies effective in routinely fixed wax embedded tissues.

Formalin fixed and paraffin wax embedded tissue from 24 cases of T-cell lymphoma diagnosed using immunocytochemistry on cryostat sections was examined using a panel of eight monoclonal and three polyclonal antisera. The monoclonal antibodies UCHL1 and MT1 proved to be comparable and reliable markers of neoplastic cells in T-cell lymphomas. The B-cell specific marker, MB1, strongly stained all cells in two cases of pleomorphic large cell T-cell lymphoma, large cells in two cases of pleomorphic mixed medium and large cell lymphoma, and isolated clusters of blast cells in four cases of T-zone and angioimmunoblastic lymphadenopathy-like T-cell lymphoma. The cells stained by MB1 expressed T suppressor/cytotoxic surface markers on frozen section. Epithelial membrane antigen, as detected by a polyclonal anti-EMA and the monoclonal antibody HMFG2, was expressed in 36% of tumours especially those of monomorphic large cell and pleomorphic large cell phenotype. Single granules or finely dispersed cytoplasmic granularity was seen in four tumours using the anti-granulocyte reagent Leu M1. Tumour cells in one case stained in a pattern identical to Reed-Sternberg cells in Hodgkin's disease. Granular alpha-1-antitrypsin staining was found in 10 cases of pleomorphic large cell and monomorphic large cell lymphoma. No staining was observed using anti-lysozyme or the monoclonal macrophage specific marker Mac411. Monomorphic and pleomorphic large cell lymphomas tended to show a common immunophenotype with the majority of cells co-expressing alpha-1-antitrypsin HLA-DR and epithelial membrane antigen. Scattered large transformed blast cells in cases of angioimmunoblastic lymphadenopathy-like T-cell lymphomas and T-zone lymphomas shared a similar immunophenotype with the large cell lymphomas. Using a panel of monoclonal antibodies effective in paraffin embedded tissue, diagnostically useful staining profiles which correlate with the morphological phenotype can be established in T-cell lymphomas.

Stain contamination and embedding in electron microscopy

Many factors (e.g., resolution of microscope, type of tissue, and preparation of sample) affect electron microscopical images and alter the amount of information that can be retrieved from a specimen. Of interest in this report are those factors associated with the evaluation of epoxy embedded tissues. In this context, informational retrieval is dependant, in part, on the ability to “see” sample detail (e.g., contrast) and, in part, on tue quality of sample preservation. Two aspects of this problem will be discussed: 1) epoxy resins and their effect on image contrast, information retrieval, and sample preservation; and 2) the interaction between some stains commonly used for enhancing contrast and information retrieval.

Prognostic importance of DNA flow cytometry in non-Hodgkin's lymphomas.

Eighty one cases of non-Hodgkin's lymphoma were examined by DNA flow cytometry, using fixed embedded histological tissue. The frequency of detection of DNA aneuploidy and the values for S phase fractions depended on the histological subtype and grade of lymphoma. Twenty two of the patients with low grade centroblastic/centrocytic non-Hodgkin's lymphoma had repeat biopsies. Eleven of these patients remained histologically and cytometrically stable, but the remaining eleven transformed into high grade non-Hodgkin's lymphoma. The mean value for the S phase fraction in the initial biopsy specimens from patients which transformed was higher than that for patients whose lymphomas remained stable (p less than 0.001). It is proposed that estimates of S phase fraction prospectively identify patients with low grade non-Hodgkin's lymphoma at risk from transformation.

Effect of tissue processing on the immunostaining of inflammatory cell surface epitopes identified by monoclonal antibodies

Optimal tissue processing conditions were defined for the immunohistochemical detection of inflammatory cell surface epitopes identified by OKT3, OKT4, OKT6, OKT8, OKIa1, OKM1, Leu7 and pan B cell antibodies. Snap-freezing in isopentan was superior to liquid nitrogen in preservation of morphology. Embedding of the tissues in Tissue-Tek II O.C.T. Compound diminished the intensity of immunostaining with all antibodies studied; however, the embedded tissues tolerated longer storage without drying. Optimal fixation with satisfactory preservation of morphology and immunogenicity was achieved with fixation of the frozen sections in acetone at 4 degrees C for 5 min. Blocking of the endogenous peroxidase with methanol-H2O2 treatment destroyed all epitopes studied except those identified with OKIa1, OKT6 and Leu7.

Pigment und Ionentransport im Vestibularorgan*

Pigments are found in various parts of the inner ear, especially in the neighbourhood of epithelia, which are supposed to be involved in the secretion and/or absorbtion of the endolymphatic fluid. Microprobe analysis (laser absorption mass micro analyzer "LAMMA" and X-ray) combined with morphological observations were performed in shock frozen, freeze-dried and plastic embedded inner ear tissue from the vestibular organ of pigmented guinea pig. Disturbance of the endolymphatic ionic composition (increased Na+) due to treatment with metabolic inhibitors (ethacrynic acid, ouabain) stimulated the migration of pigment granules and displacement of the dentritic processes of the melanocytes in a close vicinity to the presumably transporting vestibular epithelia (the dark and light cells and capillaries). The melanosomes obtained full range of metal ions that changed characteristically after treatment with metabolic inhibitors. It could be supposed that melanin presents some kind of reservoir for essential trace elements or compounds and may regulate numerous enzymatic and membrane functions by binding and releasing the metal ions.