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PubMed1984

Effects of fixation and enzymatic digestion on the immunohistochemical demonstration of laminin and fibronectin in paraffin embedded tissue.

The effects of different fixatives and enzymatic digestion procedures on the immunohistochemical demonstration of fibronectin and laminin in paraffin embedded tissues have been compared. None of the fixatives tested enabled staining of these proteins without enzymatic digestion. No intracytoplasmic laminin was found either in fixed or in fresh frozen tissue. Fixation in formol acetic acid was unsatisfactory for demonstration of fibronectin; prolonged fixation in formol sublimate was unsatisfactory for demonstration of laminin. Optimal results were achieved after fixation in routine 10% formol saline. Trypsin was completely ineffective for unmasking laminin antigens except after fixation in ethanol acetic acid; it was only partially effective for showing fibronectin antigens. The best results were obtained with protease digestion, but pepsin was an adequate, although slightly less reliable, alternative. These enzymes may be used at lower concentrations than usually recommended.

Acetates↗
PubMed2007

Treatment of tripod fracture of zygomatic bone by N-2-butyl cyanoacrylate glue fixation, and its effects on the tissues.

The standard treatment of zygomatic bone fractures is fixation by microplates or miniplates and screws today. It is very difficult to place plates and screws into thin bones or small bone fragments especially in the facial bones and bones adjacent to important structures. Cyanoacrylate is used as a hemostatic agent, an embolic agent, in retinal tears, in corneal ulcers, in fixation of mandibular osteotomies and in craniofacial surgery. N-2-Butyl cyanoacrylate is a form of cyanoacrylate which is bioabsorbable and biocompatible. It is easily applied to the tissues. We aimed to determine the effect of N-2-butyl cyanoacrylate in the fixation of displaced zygomatic bone fractures. We examined the histotoxicity and the effects on healing and foreign body reaction of N-2-butyl cyanoacrylate. Eight New Zealand white rabbits underwent zygomatic osteotomies bilaterally. The fractures on left sides of the rabbits were determined as study site and right sides as control site. Knight and North classification of zygomatic bone fractures were used and group 4 fractures were made bilaterally. Open reduction of fractures was performed bilaterally and N-2-butyl cyanoacrylate was applied only on left sides. No fixation was made on right sides representing the control group. Postoperatively in the first, second, third and fourth weeks, two rabbits were sacrificed and the fracture sites were examined macroscopically and histopathologically. In the glued study group, fixation was obtained in all cases whereas in the control group, all the fractures were seen to be displaced. Tissue reaction was similar in the study and the control groups.

Animals↗
PubMedDate not supplied

[Production and characterization of a monoclonal antibody that selectively marks the astrocyte population in the central nervous system].

The development of a monoclonal antibody that selectively labels the astrocitic population of the central nervous system in the three species tested (human, monkey and rat) is described. The tissue used as immunogen consisted in a sector of the human hippocampus (CA1) from a brain removed shortly after death. The neuropathological examination in this case revealed an congophilic angiopathy. The tissue was frozen until homogenezation and intraperitoneal injection in 8 weeks old BALB/C mice, after which lymphocytes from the spleen were fused with NS-1 myeloma cells devoid of the enzyme Hypoxantine Guanine Phosphoribosyl Transferase (HPGT (-)) following standard procedures. The resulting hybridomas were tested by ELISA assays, immunofluorescence and immunohistochemical (PAP and ABC) techniques. The monoclonal antibody obtained was identified as an IgM and the Western blot analysis indicated that it recognizes a protein of 43,000 dalton. Immunohistochemistry was performed in 30 or 50 microns sections of human temporal lobe perfused through the carotid arteries with a mixture of 4% paraformaldehyde and 0.02% picric acid. Monkey tissue (sectioned at 50 microns) was obtained from a brain with the same fixation. Rat tissue followed the same protocol, although better results were seen after shifting to 1% paraformaldehyde and 1.25% glutaraldehyde (sections were 30 microns thick). Paraffin embedded human tissue (fixed in either 10% buffered formalin or in 4% paraformaldehyde) was also tested. All immunoreactions were intensified in a solution of osmium tetroxide and thiocarbohydrazide. The monoclonal antibody gave best results at a dilution of 1:50-100, where it gave a sharp labeling of both fibrous and protoplasmic astrocytes up to the most delicate processes, both in white and the grey matter. Rat tissue fixed in only 4% paraformaldehyde resulted in less optimal labeling, mostly restricted to limiting glia and Bergmann glia. The other fixation protocol (1% paraformaldehyde and 1.25% glutaraldehyde) gave a labeling comparable to monkey and human tissue. Paraffin embedded tissue was positive and similar to that obtained in frozen tissue, only when used the 4% paraformaldehyde fixation, but not when it was fixed in 10% formalin. Therefore, this monoclonal antibody has high specificity for the astrocitic population in the nervous system of mammals and it can be useful in a variety of studies of the normal and pathological situations of the nervous system.

Aged↗
PubMedDate not supplied

Microwave techniques for diagnostic laboratories.

Microwaves (MWs) were first introduced as a method of fixation just over 20 years ago. In recent years their use has extended far beyond that of a safe, clean and rapid method of fixation of tissue blocks and large specimens, including brains. MWs accelerate the action of cross-linking fixatives and can greatly accelerate the various stages of tissue processing to produce a paraffin block in 30 min. An extensive range of ultrafast MW-stimulated special stains has been developed, and immunohistochemical procedures can be completed in 20 min by employing MWs. Cellular antigens are distinctly better preserved in tissues fixed by MWs than by conventional cross-linking fixatives. Also, the cytomorphology of cryostat sections irradiated in Wolman's solution is clearly improved. MWs can similarly be applied for fixation and staining of preparations for transmission and scanning electron microscopy, and they also greatly accelerate polymerisation of resins. In the current climate of cost containment, this wide range of applications makes the MW oven an invaluable addition to the diagnostic laboratory.

Animals↗
PubMed2006

A simple method for detection of rabies viral sequences in 16-year old archival brain specimens with one-week fixation in formalin.

Archival formalin-fixed and paraffin-embedded brain tissues are important source for diagnosis and molecular analysis. However, nucleic acids are particularly vulnerable to degradation during tissue processing. The brain cutting process usually is performed after 1 week of brain storage in formalin followed by embedding of each particular neuro-anatomical specimen in paraffin. A simple method of deparaffinization, proteinase K digestion and RNA extraction using the Boom technique to obtain rabies RNA in unbuffered, formalin-fixed and paraffin-embedded brain tissues kept at 30 degrees C for 16 years is described. Reverse transcription-polymerase chain reaction (RT-PCR) can be used to identify rabies viral N gene sequences of 150 bases in length in all patients, but not from every immunohistochemical (IHC)-positive specimen. Direct sequencing of 301bp of N gene was achieved in 4 of 7 patients. Results of sequencing a single sample of 1432 bases of N gene from a 24h processed formalin-fixed and paraffin-embedded rabies infected brain tissue after 1 month storage were in accord with those from frozen specimen analysis. It is strongly suggested that for further molecular analysis, a piece of fresh brain tissue should be saved prior to the brain sectioning process and stored no longer than 24h in formalin before embedding.

Animals↗
PubMed2003

Relative indices of water diffusion anisotropy are equivalent in live and formalin-fixed mouse brains.

Formalin fixation of tissue is a common laboratory practice. A direct comparison of diffusion tensor imaging (DTI) parameters from mouse brains before (in vivo) and after (ex vivo) formalin fixation is reported herein. Five diffusion indices were examined in a cohort of seven mice: relative anisotropy (RA), directional correlation (DC), trace (Tr(D)), trace-normalized axial diffusivity (D(axially)), and radial diffusivity (D(radially)). Seven regions of interest (ROIs), including five in white matter and two in gray matter, were selected for examination. Consistent with previous findings, a significant decrease of Tr(D) was observed for all ROIs after fixation. However, water diffusion anisotropy, as defined by the indices RA, DC, D(axially), and D(radially), remained unchanged after fixation. Thus, fixation does not appear to alter diffusion anisotropy in the mouse brain. This finding supports the utility of diffusion anisotropy analysis of fixed tissue. The combination of DTI measurements and standard histology may shed light on the microstructural determinants of diffusion anisotropy in normal and disease states.

Animals↗
PubMed2000

Optimized preembedding method improves the histologic yield of prostatic core needle biopsies.

BACKGROUND: The aim of this study was to optimize the core shape of prostatic core needle biopsies using a novel preembedding method, and to investigate the influence of the number of cores per tissue block on the histologic yield per section level. METHODS: Twenty-four core needle biopsies were taken from a fresh prostatectomy specimen, using an 18-gauge needle. Twelve core needle biopsies were conventionally fixed floating free in formalin-filled containers, whereas 12 biopsies were stretched between two nylon meshes, and placed in tissue cassettes before fixation. The total number of tissue sections per paraffin block necessary for complete workup of the tissue was determined. Using image analysis, the relation of the area of every fifth section level was calculated in relation to the total projected area of the biopsies. Both methods of tissue processing were compared for both parameters. RESULTS: In contrast to the curved biopsies after conventional processing, the optimized preembedding method generally led to stretched cores after fixation and embedding. This method led to a 100% decrease in total number of tissue sections necessary for complete workup and to higher percentages of the tissue amount per section level compared to the total projected core area. These parameters were also influenced by the number of cores per paraffin block, but to a minor degree. CONCLUSIONS: This study shows that optimized preembedding of prostatic needle biopsies improves the histologic yield per section level. The continuous occurrence of representative sections could improve diagnostic accuracy in the histological examination of prostatic core needle biopsies.

Biopsy, Needle↗
PubMed2002

Feasibility study using a natural compound (reuterin) produced by Lactobacillus reuteri in sterilizing and crosslinking biological tissues.

Bioprostheses derived from biological tissues have to be fixed and subsequently sterilized before they can be implanted in humans. Currently available crosslinking agents and sterilants used in the fixation or sterilization of biological tissues such as glutaraldehyde and formaldehyde are all highly cytotoxic, which may impair the biocompatibility of bioprostheses. Therefore, it is desirable to provide an agent suitable for use in biomedical applications that is of low cytotoxicity and may form sterile and biocompatible crosslinked products. To achieve this goal, a natural compound (reuterin), produced by Lactobacillus reuteri in the presence of glycerol, was used by our group. It is known that reuterin has antibacterial, antimycotic, and antiprotozoal activities. Additionally, as in the case with formaldehyde, reuterin may react with the free amino groups in biological tissues by using its aldehyde functional group. Therefore, it was speculated that reuterin could be used as a crosslinking agent and a sterilant for biological tissues in the same way as glutaraldehyde and formaldehyde. In the study, the production of reuterin, produced by Lactobacillus reuteri under control conditions, was reported. Preparative chromatography was used to purify reuterin. Also, the minimal inhibitory concentration and minimal bactericidal concentration of reuterin and its antimicrobial activity on a contaminated tissue were investigated. In addition, the cytotoxicity of reuterin was evaluated. Glutaraldehyde, the most commonly used sterilant in the sterilization of biological tissues, was employed as a control. Furthermore, the feasibility of using reuterin as a crosslinking agent in fixing biological tissues was studied. Fresh and the glutaraldehyde-fixed tissues were used as controls. The results obtained in the minimal inhibitory concentration and minimal bactericidal concentration studies and in the sterilization study of a contaminated tissue indicated that the antimicrobial activity of reuterin is significantly superior to its glutaraldehyde counterpart. In addition, the results obtained in the 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide assay showed that reuterin is significantly less cytotoxic than glutaraldehyde. Additionally, it was found that reuterin is an effective crosslinking agent for biological tissue fixation. The reuterin-fixed tissue had comparable free amino group content, denaturation temperature, and resistance against enzymatic degradation as the glutaraldehyde-fixed tissue. In conclusion, the results obtained in this study indicate that reuterin is an effective agent in the sterilization and fixation of biological tissues.

3T3 Cells↗
PubMed1975

[Internal fixation in the presence of soft-tissue damage in childhood (author's transl)].

Internal fixation is considered for the treatment of 10 percent at most of all fractures that occur in childhood. Only minimal fixation should be applied, to avoid the induction of an epiphyseal lesion in addition to the original fracture. Internal fixation allowing stability during weight-bearing or exercise immediately is not necessary in a child. Concomitant soft-tissue damage hardly affects internal fixation in childhood, except in the case of unstable reduction when there is extensive soft-tissue damage.

Age Factors↗
PubMed2001

Stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin).

The study was undertaken to investigate the stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin) at distinct elapsed storage durations. The glutaraldehyde-fixed counterpart was used as a control. Porcine pericardia procured from a slaughterhouse were used as raw materials. After fixation, the fixed tissues were sterilized in a graded series of ethanol solutions and thoroughly rinsed in phosphate buffered saline for 1 day, and then stored in a jar containing sterilized water. The samples were taken out and tested for their stability during the durations of 1day through 6 months after storage. The stability of each study group was tested by measuring its tensile strength, free-amino-group content, and denaturation temperature. Additionally, the cytotoxicity of each test sample and its corresponding storage solution were investigated in vitro using 3T3 fibroblasts. The results were examined using a microscope and 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that the stability of the genipin-fixed tissue during storage was superior to its glutaraldehyde-fixed counterpart. The differences in stability between the genipin- and glutaraldehyde-fixed tissues during storage may be caused by their differences in crosslinking structure. There was no apparent cytotoxicity for both the genipin-fixed tissue and its corresponding storage solution throughout the entire course of the study, whereas significant cytotoxicity was observed for both the glutaraldehyde-fixed tissue and its storage solution. However, the cytotoxicity of the glutaraldehyde-fixed tissue decreased with increasing elapsed storage duration, whereas that of its corresponding storage solution increased. This suggested that the toxic residues remaining in the glutaraldehyde-fixed tissue leached out slowly into its corresponding storage solution during the course of storage.

3T3 Cells↗
PubMed1995

Surface modification of silicone for percutaneous implantation.

In order to prevent epidermal down growth when a silicone percutaneous device was implanted, immobilization of collagen was performed onto the surface of a silicone device. The immobilization of collagen was achieved through covalent bonds between the amino groups in the collagen molecules and the carboxyl groups in poly (acrylic acid) chains grafted onto the silicone device surface. When the collagen-immobilized silicone device model was percutaneously implanted in rabbits, no sign of epidermal down growth was observed even 7 weeks after implantation, while the epidermis reached down to the deep part of the dermis as early as 3 weeks after implantation when collagen was not immobilized onto the device model surface. To have tighter fixation of the device models to the surrounding dermal tissue, the silicone device model was covered with a polyethylene sponge having an average interconnecting pore size of 150 microns. Collagen immobilization was also performed onto the sponge surface. Both the collagen-immobilized silicone device models as well as the non-treated models with polyethylene sponge were percutaneously implanted in rabbits and epidermal down growth as well as the occurrence of bacterial infection was examined. Without collagen immobilization onto the sponge surface of the device model, bacterial infection was noticed as early as 2 weeks after the implantation. The number of infected device models increased as the implantation time became longer and bacterial infection was observed in six out of seven device models at the 10th week post implantation. When the sponge surface was immobilized with collagen, bacterial infection was noticed in only one model at the 5th week after implantation. Six out of seven implanted device models with collagen immobilization were free of bacterial infection until the animals were sacrificed 30 weeks after implantation.

Animals↗
PubMed1978

Rate of DNA synthesis as an indication of drug toxicity and as a guide for scheduling cancer therapy.

The rates of incorporation of [C3H3]thymidine into the DNA of tissues of normal mice at various times after single doses of several drugs (adriamycin, vincristine, methotrexate, 5-fluorouracil [5-FU], melphalan, CCNU, and chlorozotocin) that have some clinical activity against cancer have been determined and are considered to be indicative of the "degree of normalcy" of the respective tissues at those times. These experiments show that there are some differences and some similarities in the effects of the agents upon the various tissues of the mouse. Each of the agents caused early inhibition of incorporation of [C3H3]thymidine followed by stimulation of such incorporation by intestinal mucosa and marrow, but the timing and the extents of inhibition and stimulation differed for the various agents. Except for CCNU, the preceding description would also apply to the effects upon liver and lungs, but the stimulation was delayed in comparison to that of intestinal mucosa and marrow. Early inhibition with little subsequent stimulation occurred in the spleen, kidneys, and heart. For all tissues, recovery was slower and less extensive after CCNU than after the other agents. The data obtained with the single agents were used for setting up experiments in which a single dose of one agent was followed after selected intervals of time with a single dose of a second agent. The combination of adriamycin plus 5-FU was less toxic when the two agents were given simultaneously than when the 5-FU was given 24-96 hours after the adriamycin, while for the combination of adriamycin plus CCNU there was less toxicity if the CCNU was administered 24 hours after the adriamycin rather than simultaneously with it. When a combination of a single dose of methotrexate and a single dose of 5-FU was administered to mice, the lethality was greater when the two agents were administered 120 hours apart than when the two agents were given simultaneously or were separated by 24 or 48 hours; this was true regardless of which agent was administered first. These results are consistent with what might be predicted upon the basis of the data obtained for the single agents in this study and of what is known about the mechanisms of action of these agents and of the effects of the agents in relation to cell and tissue kinetics. These results indicate that patterns of thymidine fixation by tissues following single doses of agents might be useful in scheduling multiple-drug therapy to minimize host toxicity.

Animals↗
PubMed1990

Microwave-stimulated fixation for electron microscopy using a domestic microwave oven.

We investigated microwave-stimulated fixation of tissues for transmission electron microscopy using a domestic microwave oven operating at a frequency of 2.45 GHz with an output power of 500W. Microwave-stimulated fixation, in 4% glutaraldehyde, of fresh rat kidney, liver, heart and brain tissues was compared to conventional fixation. Human renal biopsies were similarly studied. Electron microscopy showed excellent ultrastructural preservation comparable to that obtained by conventional fixation. The optimal temperature range for microwave-stimulated fixation was found to lie between 50 degrees C and 55 degrees C. Our results indicate that microwave-stimulated fixation is a rapid and reproducible technique and can be effectively applied to routine diagnostic pathology.

Animals↗
PubMed2001

Alternative fixation of bioprostheses.

The glutaraldehyde fixation technique for bioprosthetic tissue valves has been used for about 30 years. It is more or less generally recognized, however, that a solution to the ongoing problems of calcification and immunomodulated inflammation can only be achieved with the development of alternative fixation techniques that differ from fixation with glutaraldehyde. This review reports on the chemistry behind newly developed tissue-fixation processes and the preliminary results achieved with these.

Animals↗
PubMed1981

FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy.

Coupling fluorescein-isothiocyanate to dextrans (FITC-D) extends the usefulness of the dextrans as electron microscopic tracer particles by permitting preceding fluorescence stereo microscopy and high-power light microscopy of the tissue specimens. The fate of the tracer may thus be studied in vivo during the experiment, during fixation, and during the succeeding tissue processing. A study of some simple physicochemical characteristics of the tracer, and the influence, if any, of the fixing agent are also made possible. FITC-D was found to be uncharged in the pH range from 6.5 to 8.5, more rapidly precipitated by acetone than by alcohol, and to react with glutaraldehyde and osmium tetroxide in an unknown way during tissue fixation. FITC-D with molecular weights 70,000 and 150,000 showed no signs of diffusion during tissue preparation with the methods reported in the paper, whereas FITC-D 40,000 did so to a slight degree, when the tissue was kept for several days in the fixative vehicle. Securing the preservation of the lower molecular weight FITC-Ds during tissue fixation and preparation is more difficult and the described methods are not adequate. Dextrans provoke an anaphylactic reaction in most rat strains, but are well tolerated by Wistar Furth rats. The introduction of FITC into the dextran molecule might alter the biological reactions, but was also well tolerated by Wistar Furth rats. Combined fluorescence stereo microscopy, fluorescence microscopy of sections, light microscopy of strained sections and electron microscopy made it possible to follow a particular microcirculatory area, selected in vivo, to the final study in the electron microscope.

Animals↗
PubMed1997

The use of different fixatives and hydrophilic embedding media (Historesin and Unicryl) for the study of embryonic tissues.

The effects of different fixatives, dehydration procedures, and embedding media on the structural and ultrastructural preservation of young chick embryos (Hamburger and Hamilton stages 18-24) have been studied by means of light and electron microscopy techniques. Under the light microscope, the results obtained with the use of Bouin, glutaraldehyde, or glutaraldehyde-paraformaldehyde mixtures, followed by partial dehydration of the samples and the embedding with two different polar resins (Historesin and Unicryl), were compared with the results obtained using conventional paraffin-embedding methods. Cell and tissue shrinkage was determined by comparing blood cells from those embryos embedded in either of resins with those embedded in paraffin. Samples were also compared with blood smears, either methanol-fixed or unfixed, obtained from embryos at the same Hamburger and Hamilton stages. The results obtained when Unicryl and Araldite were used for electron microscopy have also been compared. When ultrastructural images from glutaraldehyde-tannic acid/osmium tetroxide fixed, Unicryl embedded samples were compared with those from araldite embedded samples, the same good results were observed with either of the resins. Araldite embedding requires a complete dehydration of the samples, while Unicryl allows the embedding of partially dehydrated embryos with optimal ultrastructural results. We suggest that these polar resins can be considered as complementary tools for embedding delicate embryonic tissues, allowing partial dehydration of the specimens with an excellent cell and tissue preservation.

Animals↗
PubMed1983

Immunohistology on formol-sublimate fixed and paraplast embedded kidney tissue with comparison to formalin fixation and to pre-embedding immunofluorescent staining.

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 micron Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.

Animals↗