Optimal methods for preparation and immunostaining of iris, ciliary body, and choroidal wholemounts.
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PURPOSE: Investigations into the biology of resident and infiltrating immune cells in the uveal tract of the rodent eye have been greatly aided by the use of tissue wholemount methods. These methods offer a number of advantages over conventional histological and frozen section techniques. The purpose of this article is to provide a detailed step by step guide to aid others who may wish to use this method. METHODS: A detailed description of whole-body perfusion fixation, dissection and isolation of the iris-ciliary body from the anterior segment and the choroid from the posterior segment is provided. In addition, the techniques used to handle whole tissue pieces during single and double immunohistochemical staining protocols, as well as the staining protocols themselves, are described. RESULTS: In refining the techniques described, the author has catalogued a number of frequent problems which compromise immunohistochemical staining results. A troubleshooting guide aimed to help identify the cause of common problems and with some suggested remedies is provided. CONCLUSIONS: Although tissue wholemounts are frequently used in retinal research, a similar approach to investigating the components of the uveal tract has only recently been applied. The methods described in this article will provide sufficient detail for other investigators to obtain maximum benefit from this alternative approach and provide an additional technique to assist in their investigations of ocular immunobiology. (+info)
Does exhaustive search for microcalcifications improve diagnostic yield in stereotactic core needle breast biopsies?
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Stereotactic core needle biopsy (SCNB) of the breast is a cost-effective alternative to needle localization biopsy for the diagnosis of mammographic calcifications. We questioned whether an exhaustive search for calcium in the small samples obtained in SCNB yields more diagnostic information than that obtained with examination of a standard number of sections. We retrospectively reviewed 168 specimens from 123 patients with mammographic calcifications, including cases in which radiographic suspicion ranged from low to high. Microcalcifications were identified on three initial levels in 112 specimens. Additional sections were examined in 50 specimens. The final diagnosis differed from the diagnosis based on three levels in 11/50 cases (22%). In 6/50 (12%), complete sectioning yielded a specific diagnosis. The increase in technical cost associated with the additional levels was 414% per case. We conclude that exhaustive searching for microcalcifications in SCNB yields a small increase in specific diagnostic information and a high technical cost. In individual cases, the additional information may be critical for appropriate patient management. (+info)
Microwave technique in histopathology and its comparison with the conventional technique.
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125 formalin fixed human tissues from different organs and 50 fresh animal tissues were taken. Each tissue piece was divided into two. Fresh animal tissues were fixed and processed in a domestic microwave oven and formalin fixed tissue were only processed in microwave oven. Simultaneous conventional processing was also carried out. Among the fresh tissues, 34 pieces were fixed in 10% formalin and 16 were stabilized in normal saline, with microwave irradiation. For histoprocessing graded ethanol (70% and absolute) for 150 tissues and graded isopropanol (70% and absolute) for 25 tissues were used for dehydration in microwave technique. Chloroform for 95 tissues, xylene for 15 tissues and isopropanol for 65 tissues were used as clearing agent in microwave technique. Liquid paraffin was impregnating agent in all 175 cases. The oven was operated at 50% power for 10 cases and 40% power for 165 cases. Recording of temperature could not be done. Regarding fixation with formalin 80% cases gave satisfactory result, while with normal saline, only 30% cases were satisfactory. Regarding dehydration with ethanol 80% were satisfactory and with isopropanol 60% were satisfactory. Regarding clearing--both chloroform and isopropyl alcohol gave satisfactory results in 80% cases but with, xylene tissues were fragmented and brittle. (+info)
A comparison of routine and rapid microwave tissue processing in a surgical pathology laboratory. Quality of histologic sections and advantages of microwave processing.
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Rapid processing of histopathologic material is becoming increasingly desirable to fulfill the needs of clinicians treating acutely ill patients. Traditional techniques for rapid processing of paraffin-embedded tissues require 4 to 5 hours, delaying treatment for some critically ill patients and requiring additional shifts of technologists in the laboratory. Microwave processing further shortens this time, allowing even more rapid histopathologic diagnosis. Few data exist comparing quality of microwave-processed tissue with that processed by more traditional techniques. We randomly selected 158 paired specimens from 111 patients. One member of the pair was processed routinely overnight, while the other was processed by the rapid microwave technique. The slides then were compared for quality of histologic preparation in a blinded fashion by 2 pathologists. Eight routinely processed specimens were judged as suboptimal, while 6 microwave-processed specimens were judged as suboptimal and 1 was considered unsatisfactory for evaluation. In the remaining cases, the material obtained by the 2 techniques was considered of identical quality. Microwave processing considerably shortens the preparation time for permanent histologic sections without a demonstrable decrease in section quality or "readability." (+info)
A retrospective analysis of background lesions and tissue accountability for male accessory sex organs in Fischer-344 rats.
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Because the paired lobes (ventral, dorsal, lateral, and anterior) of the rat prostate have not been consistently sampled in many carcinogenicity and toxicity studies, comparison among different investigations has been compromised. The lack of specific site identification for prostatic lesions further lessens the value of incidences reported. We present here the lobe-specific incidences and degree of severity of background prostatic, seminal vesicular, and ampullary glandular lesions in 1768 control Fischer-344 rats from 35 recent National Toxicology Program 2-year carcinogenicity and toxicity studies conducted in 4 laboratories. The dorsal and lateral lobes were combined and considered the dorsolateral lobe where inflammation, epithelial degeneration, mucinous cysts, and edema were observed. Inflammation in the dorsolateral lobes was significantly associated with pituitary gland adenoma whose prolactin was suggested to play an important role in pathogenesis of prostatic inflammation. Epithelial degeneration, epithelial hyperplasia, inflammation, edema, and adenoma were conspicuous in the ventral lobes. Inflammation and edema occurred in the anterior lobes (coagulating glands). Inflammation, dilatation, epithelial hyperplasia, edema, and adenoma were observed in the seminal vesicles. Inflammation was also present in the ampullary glands. We suggest an optimal embedment and trimming method in rat prostate and seminal vesicle to ensure adequate, consistent sampling. (+info)
Cell wall architecture of the elongating maize coleoptile.
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The primary walls of grasses are composed of cellulose microfibrils, glucuronoarabinoxylans (GAXs), and mixed-linkage beta-glucans, together with smaller amounts of xyloglucans, glucomannans, pectins, and a network of polyphenolic substances. Chemical imaging by Fourier transform infrared microspectroscopy revealed large differences in the distributions of many chemical species between different tissues of the maize (Zea mays) coleoptile. This was confirmed by chemical analyses of isolated outer epidermal tissues compared with mesophyll-enriched preparations. Glucomannans and esterified uronic acids were more abundant in the epidermis, whereas beta-glucans were more abundant in the mesophyll cells. The localization of beta-glucan was confirmed by immunocytochemistry in the electron microscope and quantitative biochemical assays. We used field emission scanning electron microscopy, infrared microspectroscopy, and biochemical characterization of sequentially extracted polymers to further characterize the cell wall architecture of the epidermis. Oxidation of the phenolic network followed by dilute NaOH extraction widened the pores of the wall substantially and permitted observation by scanning electron microscopy of up to six distinct microfibrillar lamellae. Sequential chemical extraction of specific polysaccharides together with enzymic digestion of beta-glucans allowed us to distinguish two distinct domains in the grass primary wall. First, a beta-glucan-enriched domain, coextensive with GAXs of low degrees of arabinosyl substitution and glucomannans, is tightly associated around microfibrils. Second, a GAX that is more highly substituted with arabinosyl residues and additional glucomannan provides an interstitial domain that interconnects the beta-glucan-coated microfibrils. Implications for current models that attempt to explain the biochemical and biophysical mechanism of wall loosening during cell growth are discussed. (+info)
Macrophage-derived chemokine expression in classical Hodgkin's lymphoma: application of tissue microarrays.
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Hodgkin's disease (HD) is a lymphoid malignancy characterized by the presence of Reed-Sternberg (RS) and Hodgkin's cells in a background of mixed inflammatory cells and stromal reaction. Studies have documented that HD is a neoplasm associated with abnormal cytokine and chemokine production. To define the expression of macrophage-derived chemokine (MDC) in HD, 57 cases (18 lymphocyte predominant, 11 mixed cellularity, 28 nodular sclerosis) were stained for MDC by immunohistochemistry and compared with reactive lymph nodes as controls. MDC was expressed by RS cells in classical HD (CHD) and showed a distinct cytoplasmic and Golgi localization. Accumulating evidence suggests that lymphocyte-predominant HD (LPHD) represents an entity distinct from CHD, with different biological properties and clinical course. On the basis of the high level of MDC staining alone, CHD could be distinguished from LPHD (P <.001), which showed only faint staining of scattered histiocytes similar to control tissues. CHD cases with high MDC mRNA levels showed high levels of MDC protein expression by immunohistochemistry (P <.001) and significant eosinophil infiltration, suggesting that MDC may represent another molecule that plays a critical role in eosinophil recruitment. We also analyzed 102 cases of non-Hodgkin's lymphoma and normal spleen, lymph node, and thymic tissue. High levels of MDC expression were specific to CHD cases because only low levels of MDC were observed in a minor subset of LPHD, NHL or normal lymphoid tissues. (+info)
In situ localization of follicular lymphoma: description and analysis by laser capture microdissection.
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From 1992 to 2000, we identified 23 lymph node biopsies with focal germinal centers (GCs) containing centrocytes staining strongly for bcl-2 protein, whereas most of the remaining lymph node showed bcl-2-negative follicular hyperplasia. We propose the designation in situ localization of follicular lymphoma (FL) for this phenomenon. In 2 additional cases, bcl-2(+) follicles with features of in situ FL were identified in association with other low-grade B-cell lymphomas. To investigate the clonality of the bcl-2(+) follicles, we performed laser capture microdissection of bcl-2(+) and bcl-2 follicles from the same lymph node in 5 cases, and analyzed them in parallel by polymerase chain reaction (PCR) amplification of immunoglobulin heavy chain (IgH) genes. In 4 of 5 cases the bcl-2(+) follicles contained monoclonal IgH gene rearrangements, whereas the bcl-2(-) GCs exhibited a polyclonal ladder. A BCL2/JH gene rearrangement was detected in 6 of 14 (43%) evaluable cases. There were 5 patients with synchronous evidence of FL at another site. There were 13 patients who, without a prior diagnosis of FL, had clinical follow-up; one developed FL in an adjacent lymph node within one year, and 2 manifested FL at 13 and 72 months, respectively. There are 10 patients who have not yet shown other evidence of FL. These results suggest that at least close to half of these cases (8/18; 44%) represent homing to and early colonization of reactive GCs by FL. Other cases might represent FL at the earliest stage of development, or a preneoplastic event, requiring a second hit for neoplastic transformation. These findings provide insight into the pathophysiology of early FL, and illustrate the utility of immunohistochemistry for early diagnosis. (+info)