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PubMed2002

Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake.

Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.

Adenosine Triphosphate↗
PubMed2002

Method for inactivating and fixing unstained smear preparations of mycobacterium tuberculosis for improved laboratory safety.

The inactivation of smears that contain Mycobacterium tuberculosis for microscopy before removal of the material from a biosafety cabinet is an important safety factor in preventing the potential transmission of tuberculosis to laboratory workers. The fixing and inactivating properties of heat flaming, 70% ethanol, and 1, 3, and 5% phenol in ethanol for smears containing M. tuberculosis were investigated. Heat flaming failed to inactivate the smear material, whereas 5% phenol in ethanol successfully fixed and inactivated all smears containing M. tuberculosis both from concentrated sputum samples and from culture material.

Bacteriological Techniques↗
PubMed1998

The effect of primary fixation with standard postfixation and the duration of hydrolysis on nuclear features in image cytometry.

The effect of three primary fixation procedures, used in the preparation of routine cytological samples: air-drying, Delaunay, and Saccomanno fixation, with postfixation in modified Böhm-Sprenger fixative, on nuclear features as a function of hydrolysis time is reported. Three different cell types: lymphatic cells (tonsil), epithelial cells (buccal mucosa) and mesenchymal cells (uterine myometrium) were used for the study. Our findings show, that generally not all features have the same plateau times as the IOD (integrated optical density), and that many features show different values depending on cell type and fixation method. It is therefore recommended that for any primary fixative used in routine clinical work and for each cell type, the hydrolysis curve for all nuclear features to be used in sample analysis should be established.

Analysis of Variance↗
PubMed1989

Optimization of immunohistochemical techniques to detect extracellular matrix proteins in fixed skin specimens.

Complete antigen visualization in the context of well-preserved tissue architecture is the goal of all immunohistochemical techniques. Frozen tissue section techniques achieve optimal antigen visualization but preserve tissue architecture poorly. On the other hand, formalin-fixed tissue section techniques preserve tissue architecture very well but result in antigen masking. Enzymatic digestion or salt extraction of formalin-fixed sections has been used to reestablish antigen expression. Recently acid-alcohol-fixed tissue has been used as a successful compromise between tissue architecture preservation and the visualization of cytoskeletal antigens. In an attempt to find an improved immunohistochemical process for non-cytoskeletal antigens, we compared avidin-biotin immunofluorescence staining in frozen, formalin-fixed, and acid-alcohol-fixed tissues. The fixed tissues were either untreated or treated with enzyme digestion or salt extraction. For this study, we examined healing cutaneous wounds in Yorkshire pigs with antibodies to fibronectin, laminin, von Willebrand factor VIII, and keratin. Although tissue architecture was poor, frozen sections provided the best antigen visualization and were therefore used as the standard for complete antigen expression. Formalin-fixed tissues had excellent tissue architecture, but most antigens were completely masked. Pre-treatment technique only partially overcame the antigen masking caused by formalin. In contrast, acid-alcohol fixation preserved tissue architecture almost as well as formalin and sometimes allowed complete antigen visualization; however, laminin and fibronectin were partially masked. Total recovery of the expression of these antigens could be obtained by pre-treating the acid-alcohol-fixed tissue with either hyaluronidase or 1 M NaCl. Therefore, acid-alcohol-fixed tissue appears best for extracellular matrix (ECM) protein immunostaining as well as for cytoskeletal staining. However, certain ECM antigens require hyaluronidase or 1 M NaCl treatment for optimal visualization.

Acetates↗
PubMed1994

Microwave-stimulated antigen retrieval is pH and temperature dependent.

The effects of the pH of solutions used in microwave-stimulated antigen retrieval were investigated for two antibodies commonly used in neurosciences, MAP-2 and SMI-32. A prolonged fixation time (longer than 1 week) prevents good immunohistochemical staining of these and many other antigens in paraffin and vibratome sections. On human brain tissue stored for 17 months in formaldehyde, the effects of different solutions during microwave treatment were compared, i.e., 4% aluminum chloride (pH 2.75), 0.1 N hydrochloric acid (pH 0.98), and citrate buffers (pH 2.5, 4.5, 6.0), together with the influence of temperature and microwave irradiation. We conclude that the pH and temperature of the solutions and the irradiation time are of great importance for the retrieval of antigens, but in a different way for the two antibodies studied. The SMI-32 staining results are optimal with citrate buffer at pH 2.5 and 2-hr irradiation at 90 degrees C, whereas MAP-2 staining results are optimal with citrate buffer at pH 4.5 and 10-min full-power irradiation. In the other conditions the staining is less intensive or less homogeneously distributed over the section.

Antibodies, Monoclonal↗
PubMed2000

Arteries of the insula.

OBJECT: The insula is located at the base of the sylvian fissure and is a potential site for pathological processes such as tumors and vascular malformations. Knowledge of insular anatomy and vascularization is essential to perform accurate microsurgical procedures in this region. METHODS: Arterial vascularization of the insula was studied in 20 human cadaver brains (40 hemispheres). The cerebral arteries were perfused with red latex to enhance their visibility, and they were dissected with the aid of an operating microscope. Arteries supplying the insula numbered an average of 96 (range 77-112). Their mean diameter measured 0.23 mm (range 0.1-0.8 mm), and the origin of each artery could be traced to the middle cerebral artery (MCA), predominantly the M2 segment. In 22 hemispheres (55%), one to six insular arteries arose from the M1 segment of the MCA and supplied the region of the limen insulae. In an additional 10 hemispheres (25%), one or two insular arteries arose from the M3 segment of the MCA and supplied the region of either the superior or inferior periinsular sulcus. The insular arteries primarily supply the insular cortex, extreme capsule, and, occasionally, the claustrum and external capsule, but not the putamen, globus pallidus, or internal capsule, which are vascularized by the lateral lenticulostriate arteries (LLAs). However, an average of 9.9 (range four-14) insular arteries in each hemisphere, mostly in the posterior insular region, were similar to perforating arteries and some of these supplied the corona radiata. Larger, more prominent insular arteries (insuloopercular arteries) were also observed (an average of 3.5 per hemisphere, range one-seven). These coursed across the surface of the insula and then looped laterally, extending branches to the medial surfaces of the opercula. CONCLUSIONS: Complete comprehension of the intricate vascularization patterns associated with the insula, as well as proficiency in insular anatomy, are prerequisites to accomplishing appropriate surgical planning and, ultimately, to completing successful exploration and removal of pathological lesions in this region.

Basal Ganglia↗
PubMed2003

A microdissection and molecular genotyping assay to confirm the identity of tissue floaters in paraffin-embedded tissue blocks.

CONTEXT: A recurring problem in surgical pathology practice is specimen mix-up and floater contamination. While many cases can be resolved histologically, a significant number remain unclear and may have serious clinical and medicolegal implications. OBJECTIVES: To design a microdissection and genotyping assay to identify contaminating floater tissues in paraffin-embedded tissues that is optimized for small samples, and to use the assay to resolve a series of clinical cases with floater tissues. MATERIALS AND METHODS: Twenty-one cases of possible tissue floater contamination in paraffin-embedded tissue blocks were included. Using 4 unstained, 4-microm-thick histologic sections, multiple sites were microdissected under direct visualization either by hand or by laser capture microdissection. Nonneoplastic and neoplastic tissues were sampled. Polymerase chain reaction was performed for a panel of 10 polymorphic microsatellite markers at 1p34, 3p26, 5q21, 9p21, 10q23, and 17p13. Allele size and content were analyzed semiquantitatively by fluorescent capillary electrophoresis, and the genotypes for the tissues in the paraffin-embedded tissue blocks were compared for identity. RESULTS: Tissue identification was successful in all cases, despite small tissue sample size and fixation effects. Comparative analysis of neoplastic tissue floaters and the presumptive source tumor was performed when possible to control for possible allelic loss or microsatellite instability. CONCLUSIONS: Microdissection and genotyping are effective and reliable means to objectively resolve problems of possible floater contamination. Even minute tissue samples provide sufficient DNA template for polymerase chain reaction microsatellite analysis. Because of the potential clinical implications of floaters, we recommend that all suspected floaters that would change a diagnosis from benign to malignant be subjected to genotyping assay to confirm the identity of the floater tissue.

Biopsy↗
PubMed1976

[Determination of the quantity of cells in different organs and tissues following alkaline dissociation].

A tissue following fixation for 10-15 days with formalin was treated with 50% KOH aqueous solution during 18 hours, which led to complete dissociation of the tissue into cells. Suspension of cells was placed into the counter of Fuchs-Rosenthal and numbers of nuclei in cells of definite tissues were counted. On preparations of the same suspension in 1000 cells distribution of the latter according to the number of nuclei was determined. Then a number of cells in 1 mg of tissue and in the organ were estimated. The count of muscular cells in the heart of a mouse showed that the relative error of the determination of the number of cells did not exceed 2.0%.

Animals↗
PubMed1993

Complete soft-tissue clubfoot release with and without internal fixation.

Two groups of patients who underwent soft-tissue release of clubfoot are compared. In group I, internal fixation (two Kirschner wires) was used for 6 weeks to retain correction of clubfoot. In group II, no internal fixation was used. Patients from the two groups were operated on by one surgeon using the same procedure. Because there was no statistically significant difference in functional result between the two treatment groups (P = .08; Mann-Whitney Test), the authors recommend avoiding internal fixation (with all the associated problems) in cases of uncomplicated clubfoot, and encourage early removal of the cast and frequent manipulations to prevent stiffness of the joints and reduce the recurrence rate.

Bone Wires↗
PubMed1986

The relevance of various tests for the study of specificity in immunocytochemical staining: a review.

Factors determining the specificity of immunocytochemical (ICC) tissue stainings as well as the various tests to study these factors are discussed. Since every specificity test only deals with particular aspects of the ICC procedure, a practical sequence of known test methods is proposed, which enables the determination of the specificity of the ICC tissue staining and, after possibly needed antiserum purification steps, may result in a monospecific staining. It is made clear that such a sequence has always to include a tissue-spectrum affinity test, in which the spectrum of tissue antigens is controlled for antibody binding. A variety of such tests, consisting of separation of tissue compounds, fixation, and ICC detection, are discussed as well as their pros and cons with respect to their predictability for the actual serum specificity in the tissue section.

Actins↗
PubMed1984

Endogenous elements in the prostate. An X-ray microanalytical study of freeze-dried frozen sections and histochemical localization of zinc by potassium pyroantimonate.

Freeze-dried frozen sections prepared from unfixed rat lateral prostate were examined by X-ray microanalysis in an attempt to establish the in vivo distribution of endogenous ions. Poor morphological resolution was a limiting factor in the analysis of subcellular regions of the tissue. Glutaraldehyde fixation prior to cryo-sectioning resulted in considerable loss of elements. The results are discussed and compared with those obtained from ultrathin sections of tissue treated with potassium pyroantimonate. Using the latter method, it was possible to demonstrate a subcellular distribution pattern for the element zinc and to correlate the metal with specific organelles. It is considered that, unlike a number of other tissues, the rat prostate does not lend itself readily to cryoultramicrotomy as a preparative regime.

Animals↗
PubMed1987

A simple staining method for chromatin in electron microscopy compatible with serial sectioning.

The study of the disposition of chromatin in the interphasic nucleus requires the combination of serial sectioning and a specific or preferential chromatin staining. The staining of chromatin with phosphotungstic acid (PTA) chromatin was originally employed on sections of glycolmethacrylate-embedded samples. As it is very difficult to obtain ribbons with this resin, we introduced a modification which consisted of staining the tissue after fixation and before dehydration, in order that epoxy resins can be applied. Several procedures were tried and the best results were attained in the following way. Standard fixation of samples no thicker than 1 mm was carried out with 2.5% glutaraldehyde at pH 7.2 for 1 or 2 h at room temperature; tissues were then rinsed three times with 0.2N HCl adjusted to pH 2.1-2.3 with 0.2N NaOH for 15 min. Staining was held with 3% W/V PTA in 1N HCl adjusted to the same pH. Samples were dehydrated in gradual ethyl alcohol concentrations and Epon-embedded. Post-staining on sections with uranyl-acetate and lead-citrate or other methods may be used to demonstrate other cell components and their relations to chromatin.

Animals↗
PubMed2001

Assessment of radical prostatectomy specimens and diagnostic reporting of pathological findings.

Proper examination of radical prostatectomy (RP) specimens from patients with prostate cancer (PCa) is critical for determining the need for adjuvant treatment and for predicting the outcome. The first step in the examination is represented by the assessment of the specimens. This includes proper fixation in formalin and whole-mount sectioning. The second step is related to the diagnostic reporting of pathological findings. This includes the evaluation of tissue features such as Gleason grade, stage and margin status. These three parameters, together with serum prostate-specific antigen (PSA) determination, are predictive factors whose prognostic role has been demonstrated in several studies.

Adenocarcinoma↗
PubMed1992

The microwave oven in temporal bone research.

The microwave oven has recently been introduced in morphological research. It can be used for virtually all procedures in histological processing; fixation, dehydration, embedding, staining, immunotechniques, and the techniques used for electron microscopy. Here we report that the microwave oven histoprocedures also have several advantages in temporal bone research on animals. The microwave oven has been tested for (i) fixation, (ii) decalcification, (ii) staining of semithin plastic sections. The microwave oven procedures are time-saving and results in an excellent preservation of the tissues. The microwave histoprocessing should also be applicable to temporal bone research in human.

Animals↗
PubMed1989

[In situ hybridization: principles and applications].

In situ hybridization is a molecular biological technique recently introduced in histology. Its principle consists of forming stable nucleic hybrids in tissues or cells. In this review, the main steps of the technique are discussed in regard to the preparation of probes and their labelling, the fixation of tissues and cells and their permeabilization in order to facilitate the penetration of labelled probes. Conditions of hybridization and requisite control reactions are also analysed. Quantification possible when radioactive probes are used is detailed. The three current applications of in situ hybridization, the localization of a gene on a chromosome, the demonstration of viral genomes in cells and the investigation of messenger RNAs coding for a determined protein, are illustrated with specific examples.

Animals↗
PubMed1991

The small pin circular fixator for distal tibial pilon fractures with soft tissue compromise.

A Monticelli-Spinelli small pin circular external fixator was used in combination with closed reduction or a limited open reduction internal fixation in five cases in an attempt to salvage a satisfactory result in distal tibia pilon fractures when associated soft tissue compromise prevented standard fixation with plates and screws. The small pin fixator enhances the ability to perform a closed reduction through a technique that uses distraction with pins in the tibia and calcaneus, combined with correction of angulation by tensioning wires with a stop nut. Small pin stabilization of these comminuted fractures allows early patient mobilization. The small diameter pins support the soft cancellous bone fragments. This technique attempts to combine the benefits of traction, external fixation, and limited internal fixation. We recommend this technique as a salvage procedure when plates and screws are contraindicated because of poor bone and soft tissue conditions.

Adult↗