Differences In Staining Research Articles

P3.03-016 Association between the Stainability of the Neurofibromatosis Type 2 Gene-Related Protein Merlin and the Tumor Properties of Mesotheliomas: Topic: Mesothelioma Transitional

Mutations in the genes cyclin-dependent kinase inhibitor 2A (CDKN2A), BRCA-1 associated protein 1 (BAP1), and neurofibromatosis type 2 (NF2) are observed in malignant pleural mesothelioma (MPM). We observed biallelic BAP1 alterations in 61% of MPMs and found that mutations are particularly frequent in epithelioid-type malignant mesotheliomas (Cancer Sci, 103:868-74, 2012). In addition, Loss-of-function mutations in NF2 are relatively frequent (40%) in MPMs have indicated. Merlin is a tumor suppressor protein coded by NF2; both merlin and the ezrin/radixin/moesin proteins are associated with suppression of invasion and metastasis of tumor cells. In this study, we examined the association between stainability of merlin and the tumor properties of MPM. Following definitive histological diagnoses of 35 cases of MPM (epithelial: n=31, biphasic: n=2, desmoplastic: n=2), we conducted immunohistochemical staining for merlin in thin sections of paraffin-embedded tumor tissue. Stainability was assessed with H-scores. The clinical stage of MPM was defined at the time near the tumor tissue harvesting time; the association between the clinical stage and therapeutic outcomes was assessed based on the outcomes of first-line chemotherapy with cisplatin (CDDP) or carboplatin(CBDCA) plus pemetrexed (PEM). The anti-merlin antibody used was LS-B394 (LSBio, Seattle, Washington, USA). 1) Seven MPMs (20%) were negative or weakly positive for merlin (H-score 0-30); of these seven MPMs, one was desmoplastic, while six were epithelial. Six MPMs were strongly positive for merlin (H-score ≥250); all six of these MPMs were epithelial. 2) No difference in merlin stainability was observed between epithelial (n=31) and non-epithelial (n=4) MPMs. Similarly, on examination of the association between stainability and IMIG staging, no differences in stainability were observed between stages 1 to 4. 3) No differences in stainability were observed between the first-line chemotherapy partial response group and progressive disease group. Loss-of-function mutations in the tumor suppressor gene NF2 lead to enhanced expression of focal adhesion kinase; although these mutations are demonstrated in decreased normal expression of the tumor suppressor protein merlin, no trend was observed in the morphological differentiation patterns of MPM. In addition, although the ratio of cells with high aldehyde dehydrogenase enzymatic activity is increased by CDDP/PEM treatment, we observed no association between CDDP/PEM sensitivity and merlin stainability. The association between NF2/merlin and tumor properties must be studied in more cases.

Pathology workflow and the integration of image analysis

In today’s pathology lab, understanding and optimizing the workfl ow becomes more and more important. Th e integration of image analysis into the workfl ow requires special considerations for generating correct results. Currently, image analysis in pathology is used primarily for the quantifi cation of IHC slides like Her2. Pathology laboratories can choose from a number of image analysis systems which scan complete slides at different magnifi cations and generate virtual slides for review by pathologists. Pathologists can mark regions of interest on virtual slides for quantifi cation. Specifi c algorithms can then be applied to each region, generating results/scores that are calculated for these regions. Th e resulting data is then, together with pre-selected images, compiled into a customizable report. Th ere are four key components for image analysis: • Staining quality • Image quality • Algorithm • Region selection Staining quality can be improved through the use of automated staining with defi ned staining protocols. Additionally, linking specifi c pre-treatment, antibody, and staining protocols to an image algorithm is key for consistent results. Having consistent staining is necessary for successful image analysis because diff erences in staining lead to diff erences in acquired digital images thereby aff ecting image analysis. Image quality can be achieved through automation and calibration. Algorithms need to be specifi c to the antibody used to generate the appropriate results. Region selection relies on the pathologist’s expertise. Th us, image analysis is used as an aid to the pathologist. A key component to image analysis is the integration of the image analysis system into the overall lab workfl ow. For example, Dako Link is used to connect diff erent instruments in the IHC laboratory and to capture data from diff erent steps in the slide preparation process, which are stored and can be retrieved. In this workfl ow, the fi rst step is accessioning, where case information is captured together with the requested staining protocol. Th e automated staining instrument uses this information (including the antibody and the staining protocol) to stain the slide. Th e actual information for a specifi c slide is then stored. Once the slides are stained and ready, they are loaded onto the ACIS® III, which accesses the Dako Link database and retrieves information about the slide. Th e information is then used to select the appropriate algorithm, which will be used to process regions on the slide. By linking automated staining systems with image analysis systems, the user is not required to re-enter information and therefore the opportunity for errors is lowered, while at the same time it is guaranteed that the correct algorithm is used for the slide. Th is concept could be used to connect other image analysis systems to laboratory workfl ow, improving overall workfl ow and reducing the number of opportunities for transcription and other errors. In the future, digital pathology will include digitization of the complete workfl ow. Information will be generated in the lab, including staining information and digitization of the slide and the process will be completed by image analysis.

Structural modifications induced in Chinese hamster V79--E chromosomes by prefixation treatment in vitro with the AT-specific agents netropsin, distamycin A, and Hoechst 33258.

Prefixation treatment of Chinese hamster V79-E cells with the AT-specific agents netropsin, distamycin A and Hoechst 33258 produced a dose- and time-dependent alteration of chromosome morphology. Compared to control cultures the chromosomes appear less condensed and revealed, after Giemsa staining, a non-random pattern of light and dark segments. It was found that the induced banding pattern corresponded for the three agents tested. Some of the darkly stained regions coincided with the extracentromeric C-bands seen in the karyotype of this cell strain, suggesting these areas to be GC-rich heterochromatin. The binding specifity and the mode of action of netropsin, distamycin A and Hoechst 33258 in DNA and chromatin suggest that the structural modifications induced depend on the amount of AT-base clusters in specific regions. Differential deprivation of chromosomal proteins might also contribute to the observed differences in stainability of chromosome segments. Attempts to induce elongations or banding patterns by prefixation treatment with the GC-specific agents actinomycin D and oligomycin were unsuccessful.

Regional Difference in Stainability with Calcium-Sensitive Acetoxymethyl-Ester Probes in Mouse Brain Slices

Loading neurons with membrane permeable Ca2+ indicators is a core experimental procedure in functional multineuron Ca2+ imaging (fMCI), an optical technique for monitoring multiple neuronal activities. Although fMCI has been applied to several brain networks, including cerebral cortex, hippocampus, and cerebellum, no studies have systematically addressed the dye-loading efficiency in different brain regions. Here, we describe the stainability of Oregon Green 488 BAPTA-1AM in mouse acute brain slice preparations. The data are suggestive of the potential usability of fMCI in many brain regions, including olfactory bulb, thalamus, dentate gyrus, habenular nucleus, and pons.

Kinetics of indocyanine green dye after intraocular surgeries using indocyanine green staining.

To determine the elimination kinetics of indocyanine green (ICG) after intraocular operations using ICG staining. Intraocular fluorescence of ICG was determined using the ICG angiographic mode of a scanning laser ophthalmoscope (in vivo) and fluorescence microscopy (in vitro) after circular curvilinear capsulorhexis with ICG staining during cataract surgery and internal limiting membrane (ILM) peeling with ICG staining during macular hole surgery. We studied 9 eyes of 7 patients with white cataracts and 14 eyes of 14 patients with idiopathic macular holes. Scanning laser ophthalmoscopy revealed fluorescence in the anterior segment of patients with cataracts on the first postoperative day, and fluorescence remained for a mean +/- SD of 6.0 +/- 2.2 days postoperatively. Scanning laser ophthalmoscopy also revealed fluorescence in the posterior pole of patients with macular holes, and it remained for a mean +/- SD of 2.7 +/- 1.4 months postoperatively. Fluorescence microscopy showed fluorescence of the entire tissues, suggesting that ICG had stained not only the surface of the membranes but had also entered them. In both operations, visual outcomes were not significantly different from the results obtained without ICG. Because entire tissues were stained, the differences in ICG kinetics might also be caused by factors other than differences in stainability, such as the environment surrounding the tissues or molecular structural differences between the lens capsule and the ILM. Although we found complete disappearance of fluorescence and good functional recovery, the longer resident time of the dye after macular hole surgery may suggest a potential risk to intraocular tissues.

Myxosporeans and Their Hyperparasitic Microsporeans in the Intestine of Emaciated Tiger Puffer.

Three myxosporeans and 2 hyperparasitic microsporeans were found in the intestine of emaciated cultured tiger puffer Takifugu rubripes in Kyushu district of Japan. Morphology of the 3 myxosporeans were examined and described as Myxidium fugu n. sp., Leptotheca fugu n. sp. and unidentified Myxidium sp. Spores of M. fugu n. sp. having 2 club-shaped polar capsules were bean-shaped, 14.0 (13.5-15.5) μm in length and 9.0 (8.0-10.0) μm in width. Spores of L. fugu n.sp. having 2 spherical polar capsules were trapezium-shaped, 9.0 (8.3-9.5) μm in length and 14.0 (13.0-15.0) μm in thickness. Their piscine intestinal developmental stages were illustrated and arranged in a hypothetical developmental sequence. Plasmodia of M.fugu n. sp. were located on the epithelium of intestine, whereas those of L. fugu n. sp. and Myxidium sp. were found intercellularly in the intestinal epithelium. Only a few mature spores could be observed in these myxosporeans, but the differences in stainability by Diff-Quik and Uvitex 2B and in location of development made it possible to differentiate the species. Two hyperparasitic microsporeans were often found in the plasmodia of M. fugu n. sp. and L. fugu n. sp, but their pathogenic effects to host myxosporeans were unclarified.

Expression of heat shock proteins in squamous cell carcinoma of the tongue: an immunohistochemical study.

Twenty-four specimens of squamous cell carcinoma of the tongue were immunostained for heat shock proteins (HSPs) to reveal differences in stainability among normal epithelium, dysplasia and carcinoma and to clarify the prognostic significance of HSPs in comparison with survival period, clinical stage, lymph node metastasis, histological grade, and p53 immunostaining. Normal epithelium was positively stained in the suprabasal layer for HSP60 and HSP70, but was negative for HSP27 and HSP90. Dysplastic lesions were positive for HSP27, HSP70 and HSP90, but stained variously for HSP60. In squamous cell carcinoma, the cytoplasm of suprabasal tumor cells was often positive for HSP27 and HSP90 (18/24, 17/24, respectively). Although HSP immunohistochemistry has revealed changes in HSP expression during tumorigenesis of squamous epithelium of the tongue, there was no correlation between HSP staining and survival period, stage, lymph node metastasis, histological grade or p53 immunostaining.

Histochemistry and immunohistochemistry of extracellular matrix of rat fetal Ductus arteriosus during the indomethacin-induced constriction.

Administration of anti-inflammatory non-steroidal drugs to pregnant animals causes constriction of the fetal ductus arteriosus (DA). To clarify the mechanism of the indomethacin-induced constriction of the rat fetal DA, extracellular matrix (ECM) components were observed by immunohistochemical techniques with antibodies 2B1, 6B6, 3B3, 9A2 and others to various kinds of proteoglycans (PGs). In the 10mg/kg group, the large PG revealed with antibody 2B1 was increased in the inner and outer media at constriction, and chondroitin 6-sulfate PG was strongly immunostained with antibody 3B3 as granular substances in the outer media, although no difference in stainability of chondroitin 4-sulfate PG revealed with the antibody 9A2 was observed between experimental and control groups. This suggests that the cells composing the fetal DA showed a tendency of proliferation and resultant production and aggregation of large PG with chondroitin 6-sulfate chains during constriction induced by indomethacin-treatment.

On the relationship of amino acid composition to silver staining of proteins in electrophoresis gels: II. Peptide sequence analysis

The quantification of proteins in silver-stained electrophoresis gels has been limited by the differences in "stainability" of different proteins. Despite efforts by many researchers, the precise basis of the reaction between silver reagents and polypeptides is still unclear, and, depending on the formulation, may even differ. We have tested the hypothesis that differences in stainability among proteins can be attributed to differences in di- or tripeptide composition. The results indicate that some order of protein structure other than short peptides accounts for the staining differences observed.

Hepatocellular Carcinoma Presenting Well Differentiated, Normotrabecular Patterns in Peripheral or Metastatic Loci

In a series of 103 surgically resected hepatocellular carcinomas (HCCs), the tumor boundaries were rather clearly defined in 85 cases (82.5%). Twelve tumors had an ill-defined margin because adjacent pseudolobules were replaced by HCC with a well differentiated growth pattern (group A1). This peripheral pattern was unrelated to the predominant histologic type of the major tumor nodules. Despite its close resemblance to normal hepatic parenchyma, we were able to determine the area of the normotrabecular HCC on the basis of, differences in stain-ability, variable nuclear crowding and frequent microacinar formation. Moreover, scattered occurrence of nuclear atypia and midtrabecular conversion were also observed within the normotrabecular HCC. In an additional 6 cases, a band-like zone of normotrabecular HCC was evident peripherally around the primary lesion (group A2). Fourteen other cases (13.6%) had intrahepatic metastatic lesions formed either within the portal vein or in pseudolobules. Of these, 5 cases showed intrapseudolobular metastases with a normotrabecular pattern (group B). For precise evaluation of surgical HCC specimens, it seems to be particularly important to realize that HCCs sometimes have a highly differentiated appearance in a locus of peripheral invasion as well as intrahepatic metastasis.

Ultrastructural identification and clinical significance of light microscopic giant mitochondria in alcoholic liver injuries.

The identification and clinical significance of light microscopic giant mitochondria (GM) were investigated in liver biopsy specimens of 60 alcoholics. By light microscopic examinations using the same section and neighboring sections, we suggest that light microscopic GM correspond to the crystalloid bodies (CB) detected on ultrastructural observation. The reasons are as follows: (1) difference in stainability between eosinophilic light microscopic GM and acidbasophilic mitochondria; (2) similarity in morphological features and density of the round- and cigar-shaped types at the light microscopic level to those of CB at the electron microscopic level; (3) similarity of the area occupied in the hepatic cytoplasm at both light and electron microscopic levels; (4) the crystalline structure of CB in compatible with the subtype of Mallory body (MB); (5) a description of CB has been made at the electron microscopic level, although not yet at the light microscopic level. Moreover, we clinically observed that light microscopic GM seldom appeared either in early fatty liver of cases aged 35 or less or the late macronodular liver cirrhosis stage of alcoholic liver injuries while they were frequently recognized during the acute aggravation phase of the chronic stage (GPT: p less than 0.05). Furthermore, if one was to assume that MB is a change accompanying necrosis of the liver cells, the light microscopic GM might be a change accompanying degeneration of the cytoplasmic organelles.

Immunohistochemical and biochemical study of alkaline phosphatase in bone tumors and tumorous conditions - With special notes on giant cells.

Immunohistochemical and biochemical investigations were carried out on alkaline phosphatase (ALPase) in 139 patients with bone tumors and tumorous conditions.Biochemically, preoperative serum ALPase level was significantly higher in osteosarcoma and chondrosarcoma, and was significantly reduced postoperatively in osteosarcoma. The ALPase activity in the tumor tissue extract was correlated with the serum ALPase level. All these isoenzymes were considered to be bone type ALPases (ALP3).Liver/bone type ALPase immunostaining was carried out to clarify the following points: 1) Localization of ALPase antigen, 2) differences in stainability between giant cells and small mononuclear cells and 3) relationship of ALPase with calcification by double staining with von Kossa stain. The results were similar to those of enzyme-histochemistry except that stromal cells in the giant cell tumor of bone (GCT) and enchondroma cells were positive for ALPase. Tumorous giant cells were positive in osteosarcoma, but not in GCT and chondroblastoma. Non-tumorous giant cells were all negative. The staining pattern was different between giant cells and stromal cells in GCT, suggesting differences in the origin, differentiation, or intracellular condition between these cells. Differences were present between calcification mediated by osteoid and that in cartilaginous tumors.

Immunohistochemical evaluation of keratin and involucrin distribution in Bowen's disease.

Specimens from patients with Bowen's disease were studied for keratin and involucrin distribution as determined by antibodies polyclonal (TK), and monoclonal (KL1 and PKK1) against keratin, and polyclonal antibodies against involucrin, respectively. Squamous cells in normal skin were positive for TK detectable keratins throughout the epidermis, while the spinous cells were strongly positive for KL1 detectable ones. Basal cells were negative for KL1, but characteristically strongly positive for PKK1. Normal epidermis showed marked staining for involucrin in upper spinous and granular cell layers. Specimens from patients with Bowen's disease exhibited irregular expression of antigen by the three types of anti-keratin antibodies with a loss of the regular or zonal distribution pattern as found in the normal epidermis. The lesions also showed negative staining for involucrin. Thus, affected epithelial cells in Bowen's disease showed marked differences in stainability with respect to present four immunoreagents.

Recognition of cells in mitosis by flow cytofluormetry.

Cells in mitosis may be distinguished from interphase cells based on difference in chromatin structure as revealed by two different methods of staining with acridine orange. In the first method, cells are heated and then stained at neutral pH; the difference in stainability between mitotic and interphase cells reflects the difference in the extent of deoxyribonucleic acid denatured by heat in these cells. At a given temperature the deoxyribonucleic acid of the mitotic cell appears to be more extensively denatured than that of the interphase cell. In the second method, cells are treated with buffer at pH 1.5 (1.3 to 1.9) and then stained at pH 2.6 (2.3 to 2.9). The mechanisms involved in the differential stainability of interphase versus mitotic cells at that low pH are currently under investigation. In both methods, in addition to enumerating cells in mitosis, it is possible to quantitate cells in G1, S and G2 phases of the cell cycle.

The morphology of the sheep retina. I. The receptor cells and the pigment epithelium.

The present investigation was undertaken in order to provide a necessary ultrastructural basis for the complete interpretation of recent findings on the electrophysiology of the sheep retina and on the influence on retinal function of certain drugs and toxic substances. The sheep retina was shown to be a mixed retina with a rather large number of cones. The receptor cells were conventional in shape. Typical differences in light and electron optical density between rod and cone ellipsoids could be explained as differences in stainability of the mitochondrial content. Specific connections between the outer segment disks and the plasma membrane as well as the occurrence of phagosomes in the pigment epithelial cells are discussed in relation to the development and the renewal of the receptor outer segment. No melanin granules were seen in the pigment epithelium of the investigated specimens, which were all taken from the tapetal area of the fundus. The possible relation between lipid‐like bodies in the pigment epithelium and vitamin A metabolism is discussed.

Electron microscopic study on the newt mast cell, especially its granule-extrusion mechanism

Newt mast cells in the iris tissues were studied in Epon-embedded sections. Specific granules were composed of a matrix of different electron density and one or more dense lamellated structures. A comparison of the electron microscopic and light microscopic images of the same mast cells revealed that the lamellated structures appear as blue or dark bluish purple rods when stained with 0.05% toluidine blue; as the matrix decreases in electron density from dense to moderately dense to less dense, it becomes light-microscopically stained from dark bluish purple to purple or reddish purple, and finally to pale pink or colorless. These differences in stainability and electron density of specific granules are related to maturation of the specific granules and secretory function of the cell. From the results mentioned above and the occasional degranulation figures observed in both the mature and immature mast cells, it was suggested that extrusion or secretion of specific granules from the mast cell may be carried out in two different ways, a diacrine mechanism and a eruptocrine one.

Mast cells of the human gingiva. 2. Metachromatic cells at low pH in healthy and inflamed tissue.

By way of degranulation, the mast cells release a number of biologically active substances into the connective tissue. The present study is concerned with the relation of the gingival mast cells to the pathogenesis of gingivitis. Following fixation in Newcomer's fluid and non‐aqueous staining at pH 0.5 in acridine orange1, topographically defined zones of sections of normal and inflamed marginal gingiva, histologically classified with regard to degree of inflammation, of 56 different individuals have been studied in the fluorescence microscope. The human gingiva was found to be comparatively rich in mast cells. Three main morphological variants were observed and their topographical distribution within the tissue have been described. Marked differences in stainability between mast cells of different areas of the connective tissue have been recorded, and correlated to the state of inflammation, In spite of individual variations in mast cell density, definite patterns of frequency and distribution were observed. The number of mast cells appeared inversely correlated to the density and distribution of the inflammatory cellular exudate within the pocket area of the connective tissue. Consequently, normal gingivae generally contained more mast cells per tissue unit than the moderately inflamed tissue, which, in turn, contained more than the severely inflamed gingivae. Exceptions were found in some moderately inflamed, fibrous gingiva with evidence of strong fibroblastic activity, where there was an increased number of mast cells. On basis of the distribution, frequency and stainability of the mast cells of the gingiva, it is suggested that the mast cells of the regions adjacent to the tooth are subject to an enzymatic degranulation as elicited by products elaborated by the gingival bacterial plaque or possibly by local antigen‐antibody interactions. Substances released by degranulation may then act as mediators during the course of the inflammatory process as well as contribute to the local resistance against injury.

On the histochemical differences of aldehyd-fuchsin positive material in the fibres of the hypothalamo-hypophyseal tract of rana temporaria.

Using a modified Alcian blue/Alcian yellow method, differences in stainability in the fibres of the preoptic-hypophyseal tract of Rana temporaria were observed. Near the preoptic nucleus most of the fibres stained yellow, in the tract the fibres stained lightgreen or showed green inclusions on a yellow background, whereas at the fibre-endings a colour switch to blue-green was found. This colour switch was the most pronounced in the neural lobe, where the neurosecretion stained blue-green, whereas in the fibre-endings in the outer zone of the median eminence a colour switch to green was noticed. The results can best be explained with the assumption that a modification or transformation of the neurosecretion occurs during its transport from the preoptic nucleus to the fibre-endings. Literature related to this hypothesis is discussed.

In situ studies on the hypothalamo-neurohypophysial complex of the European eel Anguilla anguilla (L.)

Techniques for demonstrating the hypothalamo-neurohypophysial complex of the European eel ( Anguilla anguilla L.) in situ are described, and a detailed account of the morphology of the complex is presented. It is shown that up to five discrete tracts take their origin in characteristic regions of the preoptic nucleus; some of them end in a localized region of the hypothalamic floor, here called the subterminal region, while others end in the meta-adenohypophysis. Seasonal differences in stainability of the nucleus and tracts are reported and the significance of these and of the subterminal region is discussed.

Photoperiodic and osmotic influences on the ultrastructure of the hypothalamic neurosecretory system of the white-crowned sparrow, Zonotrichia leucophyrs gambelii.

An attempt was made to correlate functional changes in the neurohypophysis of the White-crowned Sparrow, Zonotrichia leucophrys gambelii, with morphologic features on the light- and electron-microscope levels. The aldehyde-fuchsin-staining anterior median eminence possesses essentially the same ultrastructural features as the non-staining posterior median eminence. The axon terminals are characterized by the presence of a large number of small vesicles (approximately 400 A in diameter) and occasional electron-dense granules. The more-or-less “depleted” anterior median eminence occasionally evident in the photosensitive bird showing testicular development is indistinguishable ultrastructurally from the more intensely staining median eminence generally characteristic of the photorefractory bird. In the median eminence, stainability and functional state do not seem to be correlated with changes in the type, size or number of vesicles. A slight increase in the number of granules was noted in the photorefractory bird but this was considered insufficient basis to account for differences in stainability.