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PubMedDate not supplied

Postembedding immunogold labeling for electron microscopy using "LR White" resin.

A method is described for performing postembedding immunogold immunocytochemistry on sections of LR White-embedded tissues. Fixation of tissue in a combination of paraformaldehyde and glutaraldehyde, or with low concentrations of glutaraldehyde followed by partial dehydration, resulted in preservation of antigenicity for a variety of proteins in different tissue samples. Good structural preservation facilitated high-resolution immunolabeling when coupled with the use of purified monoclonal antibodies. The technique is straightforward and versatile, offering the potential for many immunocytochemical applications with minimal modifications.

Animals↗
PubMed1989

Use of betapropiolactone to disinfect fresh tissue without impairing antigenicity: method applicable to human immunodeficiency virus (HIV) positive tissue.

A method for inactivating viruses in tissues is reported that does not impair the antigenicity of the Coxsackie virus or of some common tissue antigens, a common problem with standard tissue fixation methods. Tissues can be placed briefly in Betapropiolactone before cryostat sectioning without any adverse effect on preservation or antigen expression. It is suggested that use of Betapropiolactone is applicable to tissues harbouring or exposed to the human immunodeficiency virus (HIV). As betapropiolactone has been reported to be carcinogenic in rodents any potential danger can be avoided by basic simple precautions.

Antigens, Viral↗
PubMed1998

DNA damage caused by common cytological fixatives.

Tissues from nine species of plants and fungi were treated separately with eight solutions, including seven cytological fixatives (3.7% formaldehyde at pH 3.0 and 7.0, FAA at pH 3.0 and 7. 0, 1% glutaraldehyde at pH 3.0 and 7.0, and Lavdowsky's fluid at pH 3.0) and one storage buffer (SED=NaCl-EDTA-DMSO, pH 7.0). DNA from untreated tissue and SED-treated tissue was of high molecular weight (>50 kb). DNA from glutaraldehyde-treated tissues averaged 20 kb in length, while DNA from all other treatments averaged less than 8 kb in length. Each DNA was subjected to amplification using the polymerase chain reaction, followed by sequencing of 250 bp near the 3' end of the nuclear rRNA small subunit gene. Glutaraldehyde treatments (at pH 3.0 and 7.0) produced damaged bases at rates of 0. 0% to less than 0.1%. Treatments with Lavdowsky's fluid (containing mercuric chloride), FAA at pH 7.0, and SED produced rates of 0.0% to 3.6%. FAA at pH 3.0 produced rates of 7.6% to 15.6%. Nearly 100 attempts to amplify from specimens treated with 3.7% formaldehyde (at pH 3.0 and 7.0) failed, indicating extreme damage to the DNA.

Cycadopsida↗
PubMed1987

Glutaraldehyde fixation chemistry: oxygen-consuming reactions.

In tissue fixed with glutaraldehyde, dissolved O2 is rapidly consumed by two processes: residual respiration and glutaraldehyde-induced chemical uptake. The nature of the chemistry which consumes O2 during tissue fixation was investigated by studying model reactions of glutaraldehyde with amines and with homogenized tissue suspensions. The addition of glutaraldehyde to solutions of most primary amines and ammonia stimulated rapid O2 consumption. The reaction of glutaraldehyde with primary amines (e.g., 25 mM ethanolamine, glycine, or methylamine) consumed 50% of the dissolved O2 in 15 to 20 s at 37 degrees C. The initial rate of O2 uptake followed second-order kinetics with respect to the primary amine concentration. The total amount of O2 consumed was sufficient to account for the stoichiometric conversion of the primary amines to pyridines. These data are consistent with the synthesis of pyridine derivatives from glutaraldehyde-amine precursors in which the last step is an irreversible oxidation of dihydropyridines to pyridines. The addition of glutaraldehyde to homogenized muscle suspensions, in which respiration was chemically inhibited, significantly increased the rate of O2 uptake. Thus, in tissue O2 is rapidly depleted both by respiration and the chemical demands of the glutaraldehyde-amine reactions during the cross-linking process. Since these experiments were done under conditions commonly used for tissue fixation, hypoxia should be assumed to exist in biological preparations fixed with glutaraldehyde.

Aldehydes↗
PubMed2002

Does sodium fluoride in bone cement affect implant fixation? Part I: bone tissue response, implant fixation and histology in nine rabbits.

The addition of sodium fluoride to poly (methyl-methacrylate) (PMMA) bone cement may theoretically improve the fixation of joint replacement. This hypothesis was tested in an animal model using nine mature healthy lop-eared rabbits. A femoral prosthesis was inserted in both knees to resurface the patellofemoral articulation. The same acrylic cement, with and without sodium fluoride, was randomised between the two sides for prosthetic fixation. Two screw shaped implants machined from cured rods of either cement were also inserted bilaterally into the proximal tibia. Qualitative and quantitative histomorphometry of the bone tissue response surrounding the cement in the femur and the intact tibial implants revealed similar results regardless of sodium fluoride addition. Six weeks after surgery removal, torque did not significantly differ between the two sides. Our findings indicate that addition of sodium fluoride to PMMA has little effect on implant stability and bone remodeling in rabbits in the short-term.

Journal Article↗
PubMed1993

A correlation between soluble brain nitric oxide synthase and NADPH-diaphorase activity is only seen after exposure of the tissue to fixative.

In histochemical studies using fixed brain tissue, NADPH-diaphorase has been found to be colocalized with soluble nitric oxide synthase. In the present study, using fresh tissues from eight different regions of rat brain, NADPH-diaphorase activity was found mostly in the particulate fraction, whereas most of the nitric oxide synthase activity was located to the cytosolic fraction. Also, the distribution of NADPH-diaphorase activity among brain regions was different from that of nitric oxide synthase. Pretreatment of the fractions with paraformaldehyde virtually abolished the NADPH-diaphorase activity in the particulate fraction, whereas 40-60% of the NADPH-diaphorase activity remained intact in the cytosolic fraction. These results suggest that during fixation most NADPH-diaphorase activity is inactivated and only some of the NADPH-diaphorase activity associated with soluble nitric oxide synthase remains intact.

Amino Acid Oxidoreductases↗
PubMed2005

Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity.

Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson's II, 70% ethanol, UMFIX, modified Carnoy's, modified methacarn, Bouin's, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy's solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy's solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.

Animals↗
PubMed2000

In vitro surface characterization of a biological patch fixed with a naturally occurring crosslinking agent.

The study was designed to characterize the surface properties (including water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility) of a biological patch fixed with genipin, a naturally occurring crosslinking agent. Fresh and glutaraldehyde-fixed counterparts were used as controls. It was found that both glutaraldehyde and genipin are effective crosslinking agents for biological tissue fixation. Fixation of biological tissue with glutaraldehyde or genipin significantly increased its hydrophilicity and surface tension and reduced its mol ratio of adsorbed fibrinogen to adsorbed albumin as well as the amount of adhered platelet. There were no significant differences in hydrophilicity, surface tension, the mole ratio of adsorbed fibrinogen to adsorbed albumin, and the amount of platelet adhesion between the glutaraldehyde- and genipin-fixed tissues. However, the cellular compatibilities of fresh and the genipin-fixed tissues were significantly superior to the glutaraldehyde-fixed tissue.

3T3 Cells↗
PubMed1983

Powder metal-made orthopedic implants with porous surface for fixation by tissue ingrowth.

Powder metal-made orthopedic implants with a porous coating provide an effective means for implant fixation by tissue ingrowth. Additionally, the use of metal alloy powders for forming porous surfaces offers the advantage of uniform coatings on complex part shapes. With proper processing, implants with strong, porous surface layers and good substrate mechanical properties can be formed. The in vivo tests have demonstrated the need for initial implant stability to achieve bony ingrowth. Animal studies indicate an optimum pore size range of 50-400 mu, and human hip prostheses with pores in this range appear to function well. A porous surface integrated with a compatible implant design avoids undesirable bone remodeling with these types of implants.

Animals↗
PubMed1996

In vivo inhibition of transcellular water channels (aquaporin-1) during acute peritoneal dialysis in rats.

During peritoneal dialysis (PD), a major portion of the osmotically induced water transport to the peritoneum can be predicted to occur through endothelial water-selective channels. Aquaporin-1 (AQP-1) has recently been recognized as the molecular correlate to such channels. Aquaporins can be inhibited by mercurials. In the present study, HgCl2 was applied locally to the peritoneal cavity in rats after short-term tissue fixation, used to protect the tissues from HgCl2 damage. Dianeal (3.86%) was employed as dialysis fluid, 125I-albumin as an intraperitoneal volume marker, and 51Cr-EDTA (constantly infused intravenously) to assess peritoneal small-solute permeability characteristics. Immunocytochemistry and immunoelectron microscopy revealed abundant AQP-1 labeling in capillary endothelium in peritoneal tissues, representing sites for HgCl2 inhibition of water transport. HgCl2 treatment reduced water flow and inhibited the sieving of Na+ without causing any untoward changes in microvascular permeability, compared with that of fixed control rats, in which the peritoneal cavity was exposed to tissue fixation alone. In fixed control rats, the mean intraperitoneal volume (IPV) increased from 20.5 +/- 0.15 to 25.0 +/- 0.52 ml in 60 min, whereas in the HgCl2-treated rats, the increment was only from 20.7 +/- 0.23 to 23.5 +/- 0.4 ml. In fixed control rats, the dialysate Na+ fell from 135.3 +/- 0.97 to 131.3 +/- 1.72 mM, whereas in the HgCl2-treated rats the dialysate Na+ concentration remained unchanged between 0 and 40 min, further supporting that water channels had been blocked. Computer simulations of peritoneal transport were compatible with a 66% inhibition of water flow through aquaporins. The observed HgCl2 inhibition of transcellular water channels strongly indicates a critical role of aquaporins in PD and provides evidence that water channels are crucial in transendothelial water transport when driven by crystalloid osmosis.

Animals↗
PubMed1980

[Fixation of tissue specimens].

Authors studied the efficacy of various fixatives used in pathohistology. For general purposes and for impregnation techniques sodium-acetate-formalin, for the demonstration of cytoplasmic granula of endocrine cells and for staining of connective tissue elements Ort's solution and formalin with chrome-alum should be used. Demonstration of glycogen seems to be optimal after the use of the mixture of picric acid- acetic acid and alcohol as a fixative.

Fixatives↗
PubMed2006

[Molecular consequences of fixation and tissue processing: the examples of nucleic acids and proteins].

The scientific usefulness of the molecular data obtained from tissue analysis is related directly to the quality of the tissue specimen. The most ideal tissue specimen is the one that carries a complete and unaltered representation of the tissue in vivo. The aim of this review is to provide an overview of the effects of fixation and tissue processing on the content and integrity of nucleic acid and proteins.

Cryopreservation↗
PubMed2005

Investigation of a hybrid method of soft tissue graft fixation for anterior cruciate ligament reconstruction.

To increase knee stability following anterior cruciate ligament (ACL) reconstruction, development of increasingly stronger and stiffer fixation is required. This study assessed the initial pullout force, stiffness of fixation, and failure modes for a novel hybrid fixation method combining periosteal and direct fixation using porcine femoral bone. A soft tissue graft was secured by combining both an interference screw and an EndoButton (Smith and Nephew Endoscopy, Andover, MA). The results were compared with the traditional direct fixation method using a titanium interference screw. Twenty porcine hindlimbs were divided into two groups. Specimens were loaded in line with the bone tunnel on a materials testing machine. Maximum pullout force of the hybrid fixation (588+/-37 N) was significantly greater than with an interference screw alone (516+/-37 N). The stiffness of the hybrid fixation (52.1+/-12.8 N/mm) was similar to that of screw fixation (56.5+/-10.2 N/mm). Graft pullout was predominant for screw fixation, whereas a combination of graft pullout and graft failure was seen for hybrid fixation. These results indicate that initial pullout force of soft tissue grafts can be increased by using the suggested novel hybrid fixation method.

Animals↗
PubMedDate not supplied

Microwave fixation of water-cooled insect tissues for immunohistochemistry.

In this paper a new technique for microwave-accelerated tissue fixation and washing is described. The temperature of the irradiated specimens is controlled by means of a specially designed water-cooling device. Two types of fixation fluids, one of them containing rather concentrated picric acid, and a washing procedure were tested empirically on dissected nervous tissue of the Colorado potato beetle, Leptinotarsa decemlineata (Say). Entire beetles were processed in a similar way. It was shown that specimens could be irradiated as long as was required for good fixation and washing results, without the accumulation of excessive heat. No differences in morphology and immunoreactivity were observed when compared with standard immersion-fixed controls. A considerable reduction in processing time was achieved.

Acetates↗
PubMed1980

The tissue content of cyclic AMP in rats after microwave irradiation in vivo.

When anesthetized rats were exposed to microwave (power output 1.3 kw, frequency 2,450 MHz), the cyclic AMP phosphodiesterase activity showed a rapid decline in the skeletal muscle, heart, trachea and lung; no activity essentially could be detected after a 30 sec irradiation. Cyclic AMP levels in these tissues also decreased rapidly to constant values within 30 sec. Concentrations of cyclic AMP in the heart, skeletal muscle, trachea and lung measured by the tissue fixation with liquid nitrogen were approximately 3, 3, 4 and 4 times higher than those after microwave irradiation, respectively. The intravenous injection of procaterol (1 mu mol/kg), a beta 2-adrenoceptor agonist, caused a 6-fold increase in the trachea cyclic AMP level when measured after microwave fixation but only a 2-fold increase when measured after liquid nitrogen fixation. It is concluded that rapid tissue fixation by microwave irradiation may provide a useful means for obtaining reliable values of the tissue cyclic AMP content.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Fixation of canine tendons to metal.

For the purpose of developing a method to attach tendons directly to the prosthesis, canine supraspinatus tendons were attached in vitro to a metallic surface, using 3 different fixation devices: a spiked polyacetal washer (Synthes), a spiked soft tissue fixation plate (Synthes), and a newly designed Enhanced Tendon Anchor (ETA), which straddled the tendon with interlocking spikes oriented at a 20-degree angle. 2 methods were used: 1) the tendon was fixed directly to the metallic surface, or 2) a bone block containing the tendon insertion was fixed to the metallic surface. The specimens were tested for initial fixation strength in tension to failure; intact bone-muscle-tendon-bone units were used as controls. Bone block fixations were stronger than direct tendon fixations when the spiked washer or the ETA was used; this was not true of the fixation plate. The ETA was stronger than the other techniques in ultimate strength in both direct tendon fixation and bone block fixation. The soft tissue fixation plate was found to be weaker than the other techniques in bone block fixation.

Animals↗