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PubMed2005

In situ hybridization of dihydroxyacetone phosphate acyltransferase, the regulating enzyme involved in plasmalogen biosynthesis.

In situ hybridization can be carried out using different methods. The experimenter has to choose various parameters: the type of tissue fixation, the time of incubation, and the duration of the exposure time. All these parameters are determinant for the sensitivity and the resolution of this technique. This publication of technical aspects described different experiments performed for in situ hybridization on liver tissue. We may conclude on the parameters to optimize each step of the hybridization procedure. Moreover, this technique could be transposed to the brain and applied to little structures with a light expression of DHAP-AT.

Acyltransferases↗
PubMed1986

A histological comparison of phase-partition fixation with fixation in aqueous solutions.

In phase-partition fixation, tissue is immersed in a non-aqueous solvent at equilibrium with an aqueous solution of a fixing agent to minimize osmotic effects. Preservation of morphology afforded by phase-partition fixation using formalin and glutaraldehyde and several organic solvents was compared to aqueous 10% neutral buffered formalin fixation for five tissues. It was shown that phase-partition fixation can provide excellent fixation for light microscopy if the proper combinations of fixatives and solvents are used.

Animals↗
PubMed1997

A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments.

The inability to determine the precise intracellular location of non-fluorescent organic calcium chelators such as BAPTA is a persistent problem which has precluded much detailed analysis of the chelators' spatial or temporal dynamics in live cells. Similarly, following physiological experiments with fluorescent indicators like Fura-2, it has often been desirable to maintain the dye within the cell for later analysis by additional histological techniques. Based on chemical considerations, and its prior use in tissue fixation, we examined the water soluble reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a potential fixative for diverse calcium chelators. The utility of EDC, but not other common fixatives, was confirmed through electrophysiological means, through a novel ELISA, which exploits anti-BAPTA antibodies to assess the extent and kinetics of fixation; by autoradiography of neurons loaded with [14C]-BAPTA, and by immunocytochemistry and imaging of intracellular BAPTA or Calcium Green in neurons. At concentrations > 0.1 mg/ml, EDC caused virtually instantaneous, irreversible, fixation of > 95% of BAPTA free acid. Fixation of intracellular BAPTA was confirmed in hippocampal brain slices loaded with BAPTA/AM ester, and showed biphasic kinetics consistent with rapid loading and subsequent extrusion of the chelator. Immunocytochemistry on neurons microinjected with BAPTA free acid and the dye Lucifer Yellow showed BAPTA-specific staining which was distributed in the cell similarly to that of the accompanying marker dye. Application of EDC also efficiently fixed in situ analogs of BAPTA such as Calcium Green (a fluorescent Ca2+ indicator) as shown by confocal imaging of EDC-fixed brain slices loaded with this indicator. Taken together, these data show that EDC is an effective, inexpensive and versatile fixative for calcium chelators in diverse cells. The availability of a suitable fixative now makes it possible to determine the distributions of such chelators at both the light and, possibly, the electron microscope level. Two important features of EDC, arise from its specificity for free carboxyl groups. First, the ability to fix, selectively, the chelators but not their AM esters; and, second, its enormous potential as a fixative for the numerous other carboxyl-containing chelators, dyes and pH indicators currently available.

Animals↗
PubMed2002

An improved method for beta-galactosidase activity detection on muscle tissue. A light and electron microscopic study.

In the present study we describe a method for the histochemical demonstration of bacterial beta-D-galactosidase activity on skeletal muscle tissue processed for light and transmission electron microscopy. Hence allowing this enzyme to be accurately detected, bacterial beta-galactosidase expression was studied in transgenic mouse where the enzyme, with the nuclear localization signal (nlacZ), is under the transcriptional control of the striated muscle-specific promoter MLC3F. The chromogenic substrate, 5-bromo-3-indolyl-beta-D-galactopyranoside (Bluo-Gal), was used both to recognize labelled myofibers, and beta-gal positive organelles inside single myofibers. Moreover, because the preservation of enzyme is highly dependent on tissue fixation, we developed a suitable fixation solution allowing good preservation of both tissue and enzymatic activity. This was achieved by briefly fixing tissue (3 hours) in glutaraldehyde (2.5%) and paraformaldehyde (1%) in combination. This method should be taken into consideration when studying the gene therapy of muscle diseases because it is sensitive, inexpensive and not time consuming.

Animals↗
PubMed1999

Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction.

OBJECTIVES: The purpose of this study was to determine whether apoptosis is a major mechanism of cell death in patients with sepsis. The activities of caspase-3 and the antiapoptotic protein, BCL-2, were investigated also. DESIGN: A prospective study of 20 patients who died of sepsis and multiple organ dysfunction was performed. The control group of 16 patients consisted of critically ill, nonseptic patients who were evaluated either prospectively (7) or retrospectively (9). In addition, normal colon sections from seven patients who had bowel resections were included. Apoptosis was evaluated in hematoxylin and eosin-stained specimens by deoxyuridine triphosphate nick end-labeling (TUNEL) and by DNA gel electrophoresis. SETTING: Two academic medical centers. PATIENTS: Critically ill patients. MEASUREMENTS AND MAIN RESULTS: In septic patients, apoptosis was detected in diverse organs by all three methods with a predominance in lymphocytes and intestinal epithelial cells. Hematoxylin and eosin-stained specimens from septic patients demonstrated at least focal apoptosis in 56.3% of spleens, 47.1% of colons, and 27.7% of ileums. Indirect evidence of lymphocyte apoptosis in septic patients included extensive depletion of lymphocytes in white pulp and a marked lymphocytopenia in 15 of 19 patients. Hematoxylin and eosin from nonseptic patients' tissues revealed a low level of apoptosis in one patient only. The TUNEL method increased in positivity with a delay in tissue fixation and was highly positive in many tissues from both septic and nonseptic patients. Immunohistochemical staining for active caspase-3 showed a marked increase in septic vs. nonseptic patients (p < .01), with >25% to 50% of cells being positive focally in the splenic white pulp of six septic but in no nonseptic patients. CONCLUSIONS: We conclude that caspase-3-mediated apoptosis causes extensive lymphocyte apoptosis in sepsis and may contribute to the impaired immune response that characterizes the disorder.

Adult↗
PubMed1986

Intestinal mucosa is a target tissue for pancreatic polypeptide.

Studies were carried out to identify mammalian tissues capable of specifically binding mammalian pancreatic polypeptide (PP). Bovine PP (bPP) radiolabeled with 125I was purified by HPLC to yield [125I]iodo-(Tyr-27) bPP. The label was injected into three pairs of fasted littermate dogs and allowed to circulate for 5 min. One of the dogs was a control which received an excess of unlabeled porcine PP to provide competition for receptor binding. Unbound bPP was removed by perfusion with Krebs-Ringer bicarbonate and the tissue fixed in situ with Karnovsky's fixative. Tissue samples from various organs were removed, weighed, and counted. The entire gastrointestinal tract demonstrated high levels of 125I after injection of the labeled peptide. The duodenum, jejunum, ileum, and colon were the only tissues to exhibit specific binding of bPP. These tissues (mucosal and muscle layers) from experimental animals exhibited 31-76% higher binding than the corresponding tissues from the control animals. Sections of the gastrointestinal tract were scraped to separate the mucosal layer from the underlying muscle layer. The mucosal layer of the duodenum, jejunum, and ileum exhibited 145-162% increases in binding compared to the control animals. The muscle layer of these tissues demonstrated no significant increase. These findings demonstrate that mucosal layer of the small intestine is a target tissue for mammalian PP.

Animals↗
PubMed1992

The current status of cyanoacrylate and fibrin tissue adhesives.

Surgical tissue adhesives could simplify complex surgical procedures by stabilizing tissue surfaces through hemostasis, sealing wounds, and fixating tissue in areas inaccessible to suture placement. The most common surgical tissue adhesives available to the surgeon include the cyanoacrylate derivatives and the fibrin tissue adhesives. Butyl-2 cyanoacrylate (Histoacryl) is a cyanoacrylate derivative that is frequently used in Canada and Europe. This adhesive has excellent binding strength for skin closure; however, subcutaneous implantation can result in inflammation and foreign body giant cell reaction. Fibrin tissue adhesives use a fibrin clot as the binding moiety and vary in adhesive strength depending on the fibrinogen concentration of the preparation. Autologous fibrin tissue adhesives are prepared using one of several different methods, which vary in fibrinogen yield and concentration. The currently available autologous fibrin tissue adhesives demonstrate good hemostatic properties with relatively low binding strengths. This article reviews the status of cyanoacrylate adhesives and the preparation, efficacy, and clinical applications of the fibrin tissue adhesives.

Animals↗
PubMed1998

Two pore types in the inner-wall endothelium of Schlemm's canal.

PURPOSE: It has been reported that fixation conditions significantly influence the apparent pore density in the inner-wall endothelium of Schlemm's canal. In the present study, the manner in which fixation conditions affect the two subtypes of inner-wall pores, intracellular pores and intercellular (or border) pores, was investigated. METHODS: Outflow facility was measured in enucleated human eyes. Eyes were fixed under constant flow" or constant pressure conditions, microdissected to expose the inner wall of Schlemm's canal, and prepared for scanning electron microscopy. The density and diameter of the two subtypes of pores in the inner wall were measured. RESULTS: Intracellular pore density decreased with increasing postmortem time (P < 0.001) and increased with increasing volume of fixative passed through the outflow pathway (P < 0.001), whereas border pore density showed no dependence on these parameters (P > 0.25 and P > 0.15, respectively). Border pore density increased with increasing fixation pressure (P < 0.005), even though intracellular pore density showed no such dependence (P > 0.4). No correlation was found between outflow facility and the predictions of Poiseuille's law, Sampson's law, or the funneling theory for the hydraulic conductivity of the intracellular pores (P > 0.35) or the border pores (P > 0.1). CONCLUSIONS: The intracellular and border pores form two morphologically and functionally distinct populations in the inner wall of Schlemm's canal. The dependence of intracellular pore density on postmortem time and on volume of fixative passed through the outflow pathway suggests that these pores are artifacts of tissue fixation or processing conditions. That border pores do not depend on such conditions and that their presence is correlative with perfusion pressure suggests that this population may be nonartifactual. New histologic techniques for examining the inner wall of Schlemm's canal are necessary to determine the in vivo state of inner-wall pores and how they influence outflow facility.

Aged↗
PubMed1994

Glycoconjugates of the normal human colorectum: a lectin histochemical study.

Previous studies of the normal human colorectum by lectin histochemistry have used a mixture of tissues, including those derived from colons harbouring neoplasia and inflammatory bowel diseases. In the current investigation, tissues from patients without either of these conditions have been examined with a wide panel of lectins, encompassing specificities directed against both N- and O-linked sequences, using an avidin peroxidase revealing system and evaluated with a semiquantitative scoring method. The results of binding of these lectins have been compared with those seen in the resection margins of (at least 5 cm away from) colorectal carcinomas. Consistent regional variations were noted between right- and left-sided colonic tissues, with more diverse glycan structures and a greater sialyl content in the distal colon. There was evidence of graduation of formation of oligosaccharide chains in developing crypts, possibly related to the maturation and expression of glycosyl transferases responsible for the incorporation of mannose residues of N-linked oligosaccharides and of N-acetylgalactosamine and N-acetylglucosamine. Comparison with previous reports has revealed some variations, possibly related to tissue fixation and processing and to lectin concentrations employed, which raises the question of standardization of methodologies in lectin histochemical investigations.

Colon↗
PubMed1985

The action of chromium(III) in fixation of animal tissues.

Chromic salts have been studied as fixatives of mammalian tissues for light microscopy, and the binding of the metal has been examined histochemically. Tissues bind chromium(III) from aqueous solutions less acid than pH 2.5; the metal attaches mainly to collagen and basement membranes. Solutions containing chromium(III) as the only active ingredient cannot be used as fixatives because they destroy cytoplasm and cause great structural distortion. When mixed with other fixative agents, however, chromic salts can bring about considerable improvement in structural preservation. In aqueous mixtures more acid than pH 2, and in aqueous-methanolic solutions in the pH range 4.0-5.3, a chromic salt provides only a nonspecific osmotic effect: little or no metal is bound to the tissue, and an aluminium or a sodium salt can be effectively substituted. In less acid (pH 2.3-3.2) aqueous mixtures, the beneficial action of chromium(III) cannot be imitated by aluminium or sodium ions. Chromium(III) forms coordinate bonds that cross-link ionized carboxyl groups of macromolecules. The reaction occurs so slowly that such cross-links can internally strengthen a tissue only after the structure has been stabilized by rapidly acting fixative agents. Thus, a valuable future use of chromic salts may be in a post-fixation treatment to protect specimens against the adverse effects of embedding in paraffin wax. Chromium(III) might also be useful for enhancing the opacity of collagen fibrils in electron microscopy.

Aluminum↗
PubMed2000

Enzyme histochemistry on human placental trophoblasts: the effect of fixation delay on enzyme activity.

We examined the effect of time delay in tissue fixation on enzyme histochemically detectable enzyme activity and its localization in term human placental trophoblasts. Four placental enzymes, alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, and cytochrome c oxidase, were studied. A fixation delay of 15 min did not markedly alter the activity or distribution pattern of the four enzymes, excepted for a slight reduction in cytochrome c oxidase activity and the appearance of dilated endoplasmic reticula positive for glucose-6-phosphatase. A fixation delay of 60 min abolished cytochrome c oxidase activity, but the activities of the other three enzymes remained positive. When the placental tissue was stored at 4 degrees C without cutting for 24 h before fixation, cell degeneration occurred. However, alkaline phosphatase activity was still clearly demonstrable. In enzyme histochemistry, ,,immediate" fixation is superior, but even if this cannot be performed, the placentas, especially when they are from patients with rare disorders, should not be discarded. Observations made here will be useful for clinician's attempting enzyme histochemistry in organs other than the placenta.

Acid Phosphatase↗
PubMed2000

Prostate secretory granules in normal and neoplastic prostate glands: a diagnostic aid to needle biopsy.

The recent increased efficacy of diagnosing prostate carcinoma from needle biopsy can be attributed to the accelerated biopsy rate as a result of cancer screening, the greater number of core samples per set, and the increased ability to identify malignancy in progressively smaller gland foci. This improvement in histological judgement has been facilitated by more sophisticated histological criteria, which in turn depend largely on an increasing knowledge of normal histological features and their abnormal counterparts. The recent discovery of the prostate secretory granule (PSG) as part of the normal secretory mechanism has prompted our study of the PSG as a possible additional criterion for distinction between benign and malignant cells in biopsy samples. The proper delineation of PSG required glutaraldehyde-based fixation, but this change in fixation showed additional diagnostic advantages. We quantitated PSG depletion in 150 sequential core biopsy samples, evaluating benign epithelium, dysplasia (PIN), Gleason grade 3, and grade 4 carcinoma separately. Overall, 80% of carcinomas and 63% of high-grade dysplasias were markedly depleted of PSG such that no granules were seen at low-power magnification with routine haematoxylin and eosin stains. This contrast between benign and malignant epithelium was especially prominent in small carcinoma foci greatly assisting in cancer recognition. Comparison between all groups showed an advantage of glutaraldehyde-based tissue fixation over formalin fixation for prostate needle biopsy specimens, providing clear resolution of cytological detail a well as an additional histologic criterion for cancer diagnosis. HUM PATHOL 31:1515-1519.

Adenocarcinoma↗
PubMed1997

The pathologic measurement of polyp size is preferable to the endoscopic estimate.

BACKGROUND: There is no standardized technique to measure polyp size. Estimation of polyp size at endoscopy is difficult. Polyp size measurement by pathologists would seem to be an accurate alternative, but tissue fixation may alter polyp size. To evaluate methods of determining polyp size, we compared endoscopists' estimates and pathologists' measurements with measurements made by an independent examiner. METHODS: Polyps were measured by an independent investigator before and after formalin fixation. The investigator's measurement before fixation (the "gold standard") was compared with the endoscopists' estimates and the pathologists' measurements. RESULTS: Ten endoscopists removed 61 polyps with a snare in 33 patients: 82% were adenomatous and 72% were pedunculated. Mean size was 0.85 +/- 0.6 cm (SD) (range: 0.3 to 3.6 cm, 26% > or = 1 cm). Polyps remained in formalin for a mean of 239 minutes (46 to 1164 minutes). Polyps neither consistently shrank nor enlarged in formalin (maximum change +/- 0.2 cm, r = 0.99 [p < 0.001]). Interobserver agreement between pathologists' and the investigator's post-formalin measurements showed that 55 of 57 polyps (97%) were within +/- 0.3 cm. Endoscopists inaccurately estimated 11 of 56 polyps (20%) (> 0.3 cm difference from the independent examiner). Polyp size was underestimated in three instances (range 0.5 to 0.9 cm) and overestimated in eight (range 0.4 to 0.8 cm). In 5 of 11 instances (46%), this inaccuracy altered polyp size classification across the 1 cm threshold. Results were not dependent on endoscopist, histology, or polyp location. CONCLUSIONS: (1) Polyp size is not significantly affected by formalin fixation; 2) Endoscopists' estimates of polyp size are often unreliable; and, when possible, (3) Pathologists' measurements of polyp size should be used in clinical trials and in clinical practice.

Adenomatous Polyps↗
PubMed2003

Immunohistochemical comparison of anti-prion protein (PrP) antibodies in the CNS of mice infected with scrapie.

One of the pathological changes characteristic of the transmissible spongiform encephalopathies (TSEs) is the accumulation of disease-specific PrP (PrP(sc)). Immunolabeling of PrP(sc) was compared using a panel of monoclonal and polyclonal antibodies. To determine the effects of tissue fixation on immunostaining, we performed a supplementary investigation reviewing the fixatives formol saline and periodate-lysine-paraformaldehyde (PLP). The main target sites of the antibodies were similar. However the monoclonal antibodies (MAbs) 6H4, 7A12 and 8H4 revealed targeted PrP(sc) labeling with no background labeling. Although 7A12 and 8H4 did not detect early PrP deposition, we propose that during the later stages of disease 7A12 and 8H4 can be used with equal effectiveness in place of 6H4. Tissues taken during the early stages of disease that had been fixed in PLP displayed more PrP immunolabeling than tissues that had undergone formol fixation. PLP fixation on 6H4-immunostained tissue revealed interweaving granular linear PrP deposits in the hippocampus. This labeling was not observed in tissue that had undergone formol fixation, suggesting that PLP fixation might enhance the sensitivity of the immunohistochemical (IHC) detection of PrP. In the two scrapie mouse models studied here, PLP fixation and immunolabeling with the anti-PrP antibody 6H4 gave superior results.

Animals↗
PubMed1991

Assessment of antigen damage in immunohistochemistry. The vimentin internal control.

Monitoring of the quality of antigen preservation and the uniformity of tissue fixation in paraffin-embedded, formaldehyde-fixed tissues is facilitated by the routine use of an antibody to an epitope of vimentin that is partially susceptible to formaldehyde fixation. Other diagnostically useful molecules often show fixation- or processing-induced alterations that parallel those of the vimentin epitope. Thus, the use of vimentin as an internal control, or reporter molecule, and its extrapolation to other antigens allow for better interpretation of results, improved selection of fields optimal for diagnostic immunohistochemistry, and more rational application of compensatory procedures, such as protease digestion.

Animals↗
PubMed2006

Quantitative analysis of temporal artery contraction after biopsy for evaluation of giant cell arteritis.

BACKGROUND: Although the degree of contraction of temporal artery biopsy specimens after formalin fixation has been previously reported to range from 6% to 13%, the degree of contraction before fixation has not been previously studied. The aim of this study was to quantify the postexcision (prefixation) contraction of temporal artery biopsy specimens and to determine the relationship between contraction and the result of the biopsy. METHODS: All patients undergoing temporal artery biopsies from February 2003 through May 2004 were retrospectively reviewed. Contraction was determined by subtracting the in vivo and ex vivo lengths, then dividing the difference by the in vivo length to obtain a percentage of contraction. Statistical analysis was performed with the unpaired t test and Fisher exact test. RESULTS: We reviewed 37 negative and 7 positive biopsies for giant cell arteritis (GCA). For specimens positive for GCA, the mean contraction was 12%, whereas for negative specimens, mean contraction was 22%. There was a significant difference in the contraction between the specimens positive and negative for GCA (p = 0.009). The Fisher exact test revealed that GCA was positively associated with arterial contraction of less than 15% (p = 0.002). INTERPRETATION: The temporal artery can contract substantially after excision and before tissue fixation. GCA-positive specimens exhibit statistically less contractility than negative specimens.

Biopsy↗
PubMed1998

Biocompatibility study of a biological tissue fixed with a naturally occurring crosslinking reagent.

A recognized disadvantage of the currently available chemical reagents used to fix bioprostheses is the potential toxic effects a recipient may be exposed to from residues. It is therefore desirable to provide a crosslinking reagent that is of low cytotoxicity and can form stable and biocompatible crosslinked products. To achieve this goal, a naturally occurring crosslinking reagent-genipin-was used by our group to fix biological tissues. Genipin can be obtained from its parent compound geniposide, which can be isolated from the fruits of Gardenia jasminoides ELLIS. In our previous feasibility study, it was found that the cytotoxicity of genipin is significantly lower than both glutaraldehyde and an epoxy compound. Additionally, it was shown that genipin can form stable crosslinked products. The present study further investigates the biocompatibility of a genipin-fixed porcine pericardium implanted subcutaneously in a growing rat model. The fresh, glutaraldehyde-, and epoxy-fixed counterparts were used as controls. It was noted that the inflammatory reaction of the genipin-fixed tissue was significantly less than its glutaraldehyde- and epoxy-fixed counterparts. Also, the genipin-fixed tissue has tensile strength and resistance against in vivo degradation comparable to the glutaraldehyde-fixed tissue. Additionally, the calcium content of the genipin-fixed tissue measured throughout the entire course of the study was minimal. Nevertheless, further study in calcification for the genipin-fixed tissue should be conducted in a blood-contact environment. The results obtained in this subcutaneous study indicate that genipin is a promising crosslinking reagent for biological tissue fixation. However, further durability testing in vitro and in vivo are needed to determine the relative functional merits of this new crosslinker.

Animals↗
PubMed1986

The morphology of pial blood vessels of the frog, preserved by rapid freezing and freeze substitution.

Vesicular profiles in endothelial cells of frog meninges were examined in tissues preserved by rapid freezing or conventional chemical fixation. Tissues were frozen immediately after removal from the animal or after remaining in Ringers for several hours. Vesicular profiles with an average diameter of 240 nm were present in the endothelial cells in all experimental groups, demonstrating that they are not an artifact of chemical fixation or incubation in vitro. However, their concentration and morphology varied with the different preservation techniques.

Animals↗