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PubMedDate not supplied

Fixation of testicular tissue for immunohistochemical and ultrastructural examination.

The fixation of testicular tissue with glutardialdehyde destroys the antigenicity of cell epitopes in many cases. To obtain both, morphological and immunohistochemical examination, a fixation which could preserve the antigenicity and condition of the testicular structure was sought. The solution obtained was a mixture of 3.7% formalin with 0.2% glutardialdehyde and 0.05% saponin in a phosphate buffer of pH 7.4. Blocks of human tests were immersed in this fixative, embedded in Epon 812 or paraffin for conventional light and electron microscopy and immunohistochemical staining. The immunohistochemical examination was focused on the lamina propria of the human seminiferous tubules. Good preservation of the structure was observed both in light and electron microscopy. Cytological details were seen by light microscopy, especially the recognition of single tumour cells. Electron microscopically, all cells of the seminiferous tubules and the lamina propria showed a well-preserved internal structure. At the same time the peritubular cells of the lamina propria exhibited a well-expressed vimentin and desmin immunoreactivity, thus providing evidence that the corresponding epitopes retain their antigenicity under these fixation conditions. The applied fixation procedure provides comparable results in preservation of the structure to glutardialdehyde but does not destroy the antigenicity of epitopes.

Acetates↗
PubMed1991

[Microwaves in morphologic examinations: technologic progress or whim?].

Microwave-ovens shorten the time and eliminate or reduce the formaldehyde concentration in the process of tissue-fixation for light- and electron microscopy. These devices can be also applied in dehydration, and in impregnation of tissues with paraffin as well as in staining of paraffin or cryostat cut-slices. The use of microwaves improves results of immunohistochemical and ultracytochemical stainings. The best results are given by precise laboratory microwave-devices, but domestic microwave-ovens can be also used in this procedure.

Histocytochemistry↗
PubMed1994

Choice of fixation and denaturation for the triple labelling of intra-cytoplasmic antigen, bromodeoxyuridine and DNA. Application to bone marrow plasma cells.

A triple staining method of intra-cytoplasmic antigen, bromodeoxyuridine (BrdU), and DNA for fluorescence image analysis is described. Several kinds of fixation and DNA denaturation methods were tested to obtain a technique suitable for heterogeneous tissues. The model chosen was the analysis of plasma cells in bone marrow. The fluorochromes used were fluorescein isothiocyanate (FITC) for intra-cytoplasmic antigens (light chain immunoglobulins), aminomethylcoumarin acetic acid (AMCA) for BrdU, and propidium iodide (PI) for DNA. The quality of the staining was analysed according to: (1) cell morphology with a good preservation of the chromatin structure, (2) intensity of light chains and of BrdU labelling, and (3) the quality of DNA staining judged from a DNA histogram. For most of the analysed tissues, fixation with methanol followed by 0.5% paraformaldehyde and denaturation by an NaOH concentration adapted to the tissue gave good results. However, in our model fixation by methanol, followed by methanol/acetic acid and denaturation of DNA by 0.03 N NaOH was the solely satisfactory technique. A good correlation (P < 0.001) was found with the plasma cell BrdU labelling index obtained with our reference immuno-enzymatic technique. Quantification of DNA content showed a satisfactory G1 peak coefficient of variation (CV) in diploid cells and a 4C to 2C ratio equal to 2. With this technique, the nuclear and cytoplasmic structures of both myeloid cells and plasma cells were well preserved, while their sensitivity to DNA denaturation was quite different.

Antigens↗
PubMed1999

Immunohistochemical quantitation of androgen receptor expression using color video image analysis.

BACKGROUND: The immunostaining features of the androgen receptor (AR) have been studied in prostate cancer (CaP) to predict the outcome of androgen deprivation therapies. We have developed an automatic video color image analysis system for quantitation of AR expression in large samples of prostatic nuclei. METHODS: Essential criteria of immunostaining have been examined to establish a linear relationship between AR protein content and mean optical density (MOD) of the immunoperoxidase-substrate reaction product. Titration of monoclonal AR antibody, F39.4.1, and concentration and reaction time of substrate were optimized using color video image analysis. The methodology was tested twice. First, CWR22 human CaP xenograft specimens, harvested from testosterone (T)-stimulated, castrated and T-resupplemented mice, were immunostained to demonstrate the dependence of AR expression on serum androgen levels. Second, AR expression was measured in archived clinical specimens. RESULTS: In CWR22 tumor-bearing mice castrated for 6 days, AR MOD decreased to 57% of T-stimulated, intact mice. After 72 hrs of T treatment, AR MOD returned to the level measured in T-stimulated, intact mice. Sixteen radical prostatectomy specimens and 16 transurethral resection of prostate (TURP) specimens were double-labeled with F39.4.1 and anti-cytokeratin MAb (13betaE12) specific for basal epithelial cells. Benign epithelial cells exhibited lower AR MOD in prostatectomy compared to TURP specimens (P < 0.01). Differences in AR immunostaining intensity may have resulted from differences in tissue fixation of whole organ versus small tissue specimens. CONCLUSIONS: AR immunostaining can be quantitated accurately using optimized immunohistochemical criteria and video image analysis.

Animals↗
PubMed2001

Comparisons of intraosseous graft healing between the doubled flexor tendon graft and the bone-patellar tendon-bone graft in anterior cruciate ligament reconstruction.

PURPOSE: The purpose of this study was to compare intraosseous graft healing between the doubled flexor tendon (FT) graft and the bone-patellar tendon-bone (BPTB) graft in anterior cruciate ligament (ACL) reconstruction. TYPE OF STUDY: Randomized trial. METHODS: A biomechanical and histologic study was conducted with 24 adult beagle dogs. Bilateral ACL reconstructions were performed in each animal. Autogenous doubled FT and BPTB grafts were used for the left and right knees, respectively. Each end of the 2 grafts was tethered with a polyester suture to a screw post with a washer. The animals were then allowed unrestricted activities in their cages. Eight animals were killed at 3, 6, and 12 weeks, respectively. RESULTS: Histologically, the FT graft was anchored to the tunnel wall with newly formed collagen fibers resembling Sharpey's fibers by 12 weeks. These fibers were more abundant in the anterior (ventral) gap than in the posterior (dorsal) gap. In the BPTB graft, the bone plug was anchored with newly formed bone at 3 weeks, although osteocytes in the plug trabeculae were necrotic for 12 weeks. Degeneration of the tendon-bone junction in the plug progressed at 6 weeks. Tensile testing showed that the weakest site was different not only between the 2 grafts but also between the observation periods. In the FT graft, the weakest site was the graft-wall interface at 3 weeks and the intraosseously grafted tendon at 6 weeks. In the BPTB graft, the weakest site was the graft-wall interface at 3 weeks and the proximal site in the bone plug at 6 weeks. The ultimate failure load of the FT graft was significantly inferior (45.8%) to that of the BPTB graft at 3 weeks (P =.021). At 6 weeks, the load of the FT graft was 85% that of the BPTB graft without a significant difference (P =.395). CONCLUSIONS: As to the clinical relevance, the fixation device chosen for soft-tissue fixation appears to be more important than comparing it to the BPTB graft, although this has yet to be conclusively proven.

Animals↗
PubMed2004

Vacuole formation in the endothelium of rat extremity vessels depends on fixation techniques and vessel type.

Applying immersion fixation for electron microscopy, huge clear endothelial membrane-bound vacuoles of 0.1-3 microm diameter were noted in the extremity veins of Sprague-Dawley rats. Histological and electron microscopic histochemical methods were applied to determine whether they were the product of programmed cell death or any other kind of cell damage. Image analyzer was used to measure the total area of the vacuoles in the endothelium cells. Neither lipid content nor acidic phosphatase activity could be identified in the vacuoles. In saphenous and brachial veins, the vacuoles occupied 20.6 +/- 2.21% and 18 +/- 2.45% of the endothelium, respectively. Venous endothelium of two different strains of rat also contained the vacuoles. No such structures appeared in extremity arteries. Long-term tilting did not influence vacuolization. Using in vivo whole body fixation, only pinocytotic and dense microvesicles, but no vacuoles were noted. In conclusion, the clear vacuolar structures represent neither lipid inclusions nor secondary lysosomes. The method of tissue fixation is critical when venous endothelial vesicles are investigated. It is presumed that the vacuoles originated from intra- or intercellular microstructures, and that in case of the collapsible vein segments, their size is increased under the pathological-hypoxic and low-pressure-conditions of in vitro fixation.

Animals↗
PubMed2004

The effects of glyoxal fixation on the histological evaluation of breast specimens.

Glyoxal (GL), a non-formalin-containing aldehyde tissue fixative, is advocated as a superior fixative that is environmentally safe and lacks the purported carcinogenic health hazards associated with formalin use. In addition, it is advertised as requiring no antigen retrieval before immunohistochemical staining. We compared GL fixation to standard formalin fixation of breast specimens removed for microcalcifications or breast tumors. Although the hematoxylin and eosin morphology of GL-fixed and formalin-fixed tissues was equivalent, detection of microcalcifications in GL-fixed breast specimens was hampered by loss of basophilia, likely due to increased calcium solubility in glyoxal. Moreover, estrogen receptor detection in GL-fixed specimens was diminished compared to formalin and did require antigen retrieval.

Breast Diseases↗
PubMed2005

Laser microdissection and RNA analysis.

Microdissection techniques have become an important tool to link histomorphology and pathophysiological events using modern methods of molecular biology. They allow isolation of cell clusters or even single cells precisely under optical control from complex tissue structures for further analysis of DNA, RNA, and proteins. In particular, the fragile RNA molecules can be preserved during microdissection so that gene expression and regulation measurement become feasible in a cell type-specific manner within complex tissues. This report focuses on and outlines the procedures for RNA investigation, from tissue fixation, sectioning, and staining to downstream applications (RT-PCR, mRNA quantification, and mRNA preamplification). Standards for the preparation of RNA from frozen and formalin-fixed tissues are presented. Specific protocols are given for both the isolation of RNA from small numbers of cells (50 cells) as well as for larger cell numbers. While most of the procedures are identical for the microdissection systems, special features of each technique are mentioned.

Animals↗
PubMed1993

Incidence of Epstein-Barr virus in AIDS-related lymphoma specimens.

We used the polymerase chain reaction, in situ hybridization, and immunohistochemical stains against latent membrane protein, CD23, and Epstein-Barr viral nuclear antigens 1 and 2 to identify Epstein-Barr virus (EBV) in fixed and unfixed (cryopreserved) AIDS-related lymphoma (ARL) specimens. The study included 17 cases of large-cell (immunoblastic) lymphoma, 11 cases of small non-cleaved cell lymphoma, and two cases of Hodgkin's disease. The EBV DNA was more frequently detected by polymerase chain reaction in cryopreserved specimens (94%) than in fixed specimens (17%). Significantly, the immunohistochemical and in situ hybridization studies detected evidence of EBV in only a small (< 10%) subset of the cells in 27 of 30 ARL specimens. We conclude that tissue fixation reduced the ability to detect EBV in ARL by polymerase chain reaction and that EBV was detectable in only a minority of cells in most ARL tissues.

Antigens, Viral↗
PubMed2005

Fixation of soft tissue surrounded by bone with microwave irradiation: electron microscopic observation of guinea pig inner ear.

When electron microscopy is performed on organs such as the inner ear that cannot be removed immediately after decapitation of animals, it is necessary to fix the target organ or tissue by systemic or regional perfusion fixation. However, such methods of fixation can increase vascular pressure or perilymphatic pressure, making it difficult to perform precise morphological observation of the vascular endothelial cells and membranous labyrinth. We recently attempted fixation of the cochlea by microwave irradiation. Guinea pigs were decapitated. The bullas were then removed from each animal and fixed in a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde. Microwave (300 W) irradiation was then applied to the specimen for 1 minute. The fixative was immediately replaced with new fixative (4 degrees C). This sequence of manipulations was repeated 10 times, for a cumulative microwave irradiation time of 10 minutes. During the microwave irradiation period, the fixative temperature was kept at about 30 degrees C. After the last round of irradiation, the specimens were kept immersed in the fixative for 1 hour. After a small slit was created in the bone on the lateral wall of the cochlea, the specimens were post-fixed in osmic acid and embedded in Epon 812. Each specimen was cut into halves along the plane containing the modiolus of the cochlea. After the bone on the lateral wall of the cochlea was cut off under a stereoscopic microscope, ultrathin sections were prepared for observation under a transmission electron microscope. With this technique, the stria vascularis and the organ of Corti were fixed to a degree comparable to or better than that achieved with the conventional method of fixation. Fixation with microwave irradiation is relatively simple and can solve the problems associated with perfusion fixation, and thus provides an excellent means of fixation. This technique appears to be particularly promising for fixation for soft tissue surrounded by bone.

Animals↗
PubMed2006

Automated analysis of fluorescence in situ hybridization on fixed, paraffin-embedded whole tissue sections in B-cell lymphoma.

Certain recurrent cytogenetic abnormalities are diagnostic of a specific neoplasm and may portend prognosis. As conventional cytogenetics may not reveal a neoplastic clone, and unfixed material for fluorescence in situ hybridization may be unavailable, performing fluorescence in situ hybridization on fixed tissues is diagnostically and prognostically valuable. Manual interpretation of fluorescence in situ hybridization signals may be difficult on paraffin-embedded tissue sections due to truncated nuclei. Therefore, we investigated the use of an automated image acquisition and analysis system (MetaSystems) for interpretation of fluorescence in situ hybridization signals in tissue sections from dual fusion translocation probes. Three probe sets were analyzed on archival specimens with a confirmed diagnosis of mantle cell lymphoma, follicular lymphoma or Burkitt lymphoma. 100% of mantle cell lymphomas (7/7) were positive for t(11;14), 91% of follicular lymphomas (10/11) for t(14;18) and 100% of Burkitt lymphomas (9/9) for t(8;14). Successful hybridization was achieved using various tissue fixatives and fluorescence in situ hybridization interpretation was blinded with respect to the underlying diagnosis. Based on these results, automated analysis of fluorescence in situ hybridization on fixed tissues is accurate and valuable in the evaluation of B-cell lymphoma, and may provide pertinent diagnostic and prognostic information.

Burkitt Lymphoma↗
PubMed1998

Microwave procedures for electron microscopy and resin-embedded sections.

Microwaves now have well-established applications in routine light microscopy. They are employed in tissue fixation and to accelerate a wide spectrum of staining procedures. Besides producing superior preservation of cellular antigens through microwave fixation, this form of irradiation has been employed for antigen retrieval, a procedure that has been a major factor in the optimization of immunolabelling in paraffin sections and cytological preparations. A commercial tissue processor has recently been developed which employs microwaves in a markedly accelerated, one-step processing of tissue blocks, completing the procedure within a fraction of conventional times. Microwaves have also been successfully applied in a variety of molecular techniques such as in situ hybridization and polymerase chain reaction. The adoption of microwaves in electron microscopic procedures has been slower, largely because the requirement for speed in processing is not as great, except in diagnostic samples. However, as this review will show, there are equally as many innovative applications of microwaves in electron microscopy. Microwaves have been employed for rapid processing of fine needle aspiration biopsy samples, in keeping with the requirement for speed in this method of diagnosis. Ultrafast fixation of tissue samples has resulted in the better demonstration of cellular enzymes and proteins. It has been clearly shown that microwave-stimulated on grid staining in uranyl acetate and lead citrate produces more consistent results and without background precipitation. Microwaves can be used to hasten resin polymerization and exposure to microwaves results in antigen retrieval in both resin-embedded thick sections and for immuno-electron microscopy. Immunolabelling shows enhanced sensitivity and the technique is anticipated to contribute greatly to the optimization of immuno-electron microscopy. The potential for greatly accelerated preparation of samples for electron microscopy exists but is yet to be fully realized.

Image Enhancement↗
PubMed2002

Toward efficient analysis of mutations in single cells from ethanol-fixed, paraffin-embedded, and immunohistochemically stained tissues.

Only a few studies have demonstrated successful molecular analysis after whole genome amplification using single cells dissected from paraffin-embedded tissues. The results in these studies were limited by low-amplification efficiency and high rates of allele dropout. In the present study, the amplification rate using a thoroughly modified primer extension and preamplification-PCR protocol was improved significantly for single cells microdissected from paraffin-embedded and immunohistochemically stained tissues. Tissue fixation with ethanol (85%) and the addition of 0.2 mmol/L EDTA helped to achieve an amplification rate between 67% (segments 200 to 400 bp) and 72% (segments <200 bp). Normal tissue sections were immunohistochemically double stained for overabundance of p53 protein and proliferating cell nuclear antigen. Microdissection of single cells was performed with a manual micromanipulator equipped with a Tungsten needle. Sequence analysis of the TP53 gene was performed after improved primer extension preamplification-PCR and multiplex PCR from single microdissected cells. The rate of allele dropout was at least 68%. These technical advances facilitate routine mutation analysis using a single cell or a few cells microdissected from routinely processed paraffin-embedded normal and tumor tissues. Allele dropout still represents a serious problem in single-cell mutation analysis, especially in samples with limited template DNA and prone to DNA damage.

Breast↗
PubMed1992

Interleukin 1 immunoreactivity in sebaceous glands.

Interleukin 1 (IL-1) immunoreactivity in sebaceous glands was studied in paraffin sections of normal human skin. A panel of antibodies against IL-1 alpha and beta was tested using an immunoperoxidase labelling method. All the antibodies showed a similar specific labelling pattern: both glandular and ductal cells were immunoreactive for both IL-1 alpha and beta, provided optimal tissue fixation was used.

Adult↗
PubMed2002

Linear birefringence of the retinal nerve fiber layer measured in vitro with a multispectral imaging micropolarimeter.

Scanning laser polarimetry (SLP) assesses the retinal nerve fiber layer (RNFL) for glaucoma diagnosis by detecting the birefringence of the peripapillary RNFL. A detailed understanding of SLP requires an accurate value for RNFL birefringence in order to relate measured retardance to RNFL thickness, but current knowledge of this value is limited. A multispectral imaging micropolarimeter of PSC'A type was used to measure the retardance in transmission of the RNFL of isolated rat retina before (living) and after (fixed) 20 min of glutaraldehyde fixation. The thickness of the nerve fiber bundles measured was then determined histologically. As previously known from reflectance measurements, in transmission the RNFL behaved as a linear retarder. The retardance of the RNFL was constant at wavelengths from 440 to 830 nm and persisted after tissue fixation. In 37 nerve fiber bundles of 8 retinas, the average RNFL birefringence was 0.23 nm/microm before and 0.19 nm/microm after fixation, with an uncertainty of 0.01 nm/microm. The wavelength independence is consistent with a mechanism of form birefringence from thin cylindrical organelles. These results allow extrapolation of previous visible wavelength measurements to the near-infrared wavelengths used by SLP and validate the use of fixed tissue for RNFL research.

Animals↗
PubMed1992

Fixation for biological ultrastructure. I. A viscometric analysis of the interaction between glutaraldehyde and bovine serum albumin.

The increase in viscosity resulting from mixing concentrated solutions of albumin with dilute glutaraldehyde has been investigated. The viscosity changes are slow at first, very sensitive to small changes in glutaraldehyde concentration, and non-linear with time. The cross-linking of the albumin by glutaraldehyde seems to be retarded by the weak mechanical shear forces produced by the viscometer. The significance of these findings to the events in tissue fixation is discussed.

Chemical Phenomena↗
PubMed1992

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds.

Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study; I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32-34 s, final temperature between 40 degrees C and 47 degrees C.

Chlorophyta↗