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PubMedDate not supplied

A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.

Biopsy↗
PubMed1988

Antigen preservation in microwave-irradiated tissues: a comparison with formaldehyde fixation.

A wide range of immunoreactive antigens commonly investigated in diagnostic pathology were evaluated in tissues subjected to fixation by microwave irradiation in normal saline to 63 degrees C. All of the 23 antigens studied were well preserved and compared with corresponding tissues conventionally fixed by formaldehyde for periods of 16 and 18 h, respectively, immunostaining in microwave-irradiated tissues was clearly superior, being more intense and also more extensive. Proteolytic digestion was not necessary for irradiated tissues except for the staining of cytokeratins and desmin. It was also possible to stain for Leu-14 (CD22) and Leu-1 (CD5), whereas these lymphocyte antigens were lost during formaldehyde fixation. Cytomorphological details were well preserved in the rapidly-fixed irradiated tissues.

Antigens↗
PubMed2000

Treatment of isolated complex distal femoral fractures by external fixation.

Thirteen patients with isolated distal femoral fractures were treated by external fixation. There were seven males and six females with an average age of 45 years. Four were Type A3 fractures, one Type C1, five Type C2 and three Type C3 fractures. Seven of these were open. In seven cases the articular surface was first reduced and fixed. The fixation was extended across the knee to supplement the distal fixation in six severe cases. The average follow up was 30 months. There was one non-union in the study with the average time to union in the other patients being six months. Using the Mize criteria for assessing clinical results we found that nine patients obtained a good to excellent score and four were classed as failures. The average range of movement of the knee in the study was 100 degrees. Apart from the single non-union all the fractures healed and there were no other serious complications. Considering the severity of the fractures we did not find any evidence to suggest that temporary fixation of the lateral soft tissues by fixator pins was detrimental. The results suggest that external fixation may be used to treat these difficult fractures without the risk of serious complications.

Adult↗
PubMed1983

The brush border of proximal tubules of normal human kidney activates the alternative pathway of the complement system in vitro.

The aim of this investigation was to study complement fixation by normal human kidney tissue. C fixation was assessed on acetone-fixed sections of frozen human kidney. In addition, C consumption following incubation of normal fresh human serum with tubular or glomerular fractions of human kidney was measured. The results are consistent with the interpretation that the brush border of proximal tubules of human kidney activates the C system via the alternative pathway. It is suggested that this activation may occur in vivo in patients with a non-selective proteinuria.

Complement Activation↗
PubMed2000

Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs.

AIMS: To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues. METHODS: This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared. RESULTS: Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessles. CONCLUSIONS: These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur.

Animals↗
PubMed1982

[Comparative immunohistological studies of the localization of the carcinoembryonic antigen in benign and malignant breast tissue].

By means of immunoperoxidase techniques carcinoembryonic antigen (CEA) has been localized first in carcinomas of the gastrointestinal tract and then also in other malignant tissues, such as carcinomas of the female genital tract and the breast. There is still an obvious need for intensive studies of the possible effects which the initial tissue processing, i. e. different fixation procedures may have on the localization results of CEA. By using the two step HRP-labelled antibody technique, we studied comparatively the effect of the most employed fixations on tissues from patients with mammary carcinomas and benign breast lesions: 1. Formalin-fixed and paraplast-embedded tissues 2. Cryostat sections of tissues fixed in ethanol and acetone 3. Parablast embedded tissues fixed by the freeze substitution method. The latter sections were tested whether there was any difference in the CEA localization when using the immunofluorescence or immunoperoxidase staining techniques. We concluded from our studies that routinely fixed paraplast sections, stained by the two step HRP-labelled antibody sandwich technique gave sufficient results for the demonstration and localization of CEA.

Breast Neoplasms↗
PubMed2000

The pericardial bioprosthesis: altered tissue shear properties following glutaraldehyde fixation.

BACKGROUND AND AIM OF THE STUDY: Glutaraldehyde-fixed bovine pericardial tissue used in the construction of valvular bioprostheses undergoes repeated bending stress during the cardiac cycle. To bend smoothly, internal tissue shearing is required. The effect of glutaraldehyde fixation on internal shear properties of this material was examined. METHODS: Pericardium from each of 12 bovine hearts was cut into two pieces; one piece was retained as fresh tissue, the other was glutaraldehyde-fixed. Circular samples were then mounted and installed in a shear testing apparatus. For each sample, the shear stress versus shear strain characteristics were measured in circumferential and radial directions at strain rates of 1.0, 0.1 and 0.02 s(-1) while immersed in a 20 degrees C bath; similar measurements were made on six fresh and six fixed samples at 37 degrees C. In addition, the stress relaxation properties were measured by holding the tissue at maximum shear for 100 s after each of the three shear deformations, and recording force generated with time. RESULTS: The shear stress-strain test on fresh tissue (n = 12) showed non-linear behavior at the three shear rates. The shear modulus for fresh tissue increased from <1.0 kPa to 5 kPa at a shear strain approaching 1.0, and results were identical in radial or circumferential directions. For glutaraldehyde-fixed pericardium (n = 12), shear modulus increased promptly to 15-20 kPa at a strain of 0.2, and did not vary with strain rate. Shear relaxation was similar in fresh and fixed tissue. CONCLUSION: Fresh pericardium sheared easily at low shear stresses, with minimal resistance developing until the shear strain exceeded 0.5, while glutaraldehyde-fixed tissue displayed a marked resistance to shearing, with an immediate rise in shear stress at low strain. No differences were detected in shear properties between radial and circumferential directions. Such marked tissue stiffening may be a factor in collagen fiber disruption, leading to bioprosthetic heart valve failure.

Biomechanical Phenomena↗
PubMedDate not supplied

Prolonged storage of fixative for electron microscopy: effects on tissue preservation for diagnostic specimens.

Prolonged storage of Karnovsky's fixative, at both 4 and -20 degrees C, is possible in the diagnostic electron microscopy setting. Ultrastructural detail was not compromised in specimens processed in fixative that had been stored for 6 months. Evidence of smooth muscle and neuroendocrine differentiation was present in the form of actin filaments/dense bodies and neurosecretory granules, respectively. No difference in preservation was detected between specimens fixed in freshly prepared Karnovsky's fixative and fixative that had been stored at either 4 or -20 degrees C for up to 6 months. Thus, community hospitals can be provided with Karnovsky's fixative on a semiannual basis for surgical pathology specimens that may require electron microscopy for diagnosis.

Fixatives↗
PubMed1981

The management of open tibial fractures with associated soft-tissue loss: external pin fixation with early flap coverage.

Meaningful data on the management of open tibial fractures cannot be obtained unless one categorizes the injury according to fracture type, degree of soft-tissue loss, and the velocity of the injury. Treatment by converting the type III injury to a type II injury with well-vascularized soft tissue is presented. Eighteen patients with 20 type III and type IIIa wounds were treated in a prospective fashion employing a combined orthopedic and plastic surgical scheme based on the tenets of early radical debridement, a "second look" operation, muscle or muscle-skin flap cover within 5 days of injury, external pin fixation, and ambulation within the first 3 weeks of injury. All fractures united in a mean time of 4.0 months. The mean hospitalization was 4.2 weeks. There have been no chronic infection, osteomyelitis, nonunion, shortening, or tissue breakdown.

Adult↗
PubMed1989

The influence of fixatives and other variations in tissue processing on nuclear morphometric features.

The influence on the area and numerical density of nuclei was investigated in 5-mm-thick slices of guinea pig liver for different fixatives and variations in tissue processing: delay in fixation, air drying, degree of acidity of 10% formalin (= 4% formaldehyde), Bouin and mercury-formalin fixatives, acetone and ethanol dehydration and understretching and overstretching of the paraffin-embedded sections. Air drying (either forced or as a result of delayed fixation), the type of fixative and the degree of acidity affected the nuclear area. Regarding the latter, nuclear area was approximately 25% lower for pH less than or equal to 3 as compared with pH greater than 5. In comparison with the standard tissue processing used, the nuclear density was higher after all of the variations studied (air drying, acetone dehydration and fixation). These findings indicate that nuclear area, in contrast to other tissue components, is relatively insensitive to variations in tissue processing. However, it is essential to regularly measure the pH of the fixative: deviations from pH = 7 should be carefully avoided in order to keep nuclear area variations as a result of tissue processing within acceptable limits.

Acetone↗
PubMed1984

Complement fixation by pemphigus antibody. I. In vitro fixation to organ and tissue culture skin.

Although complement is often detected in the intercellular substance of pemphigus skin lesions, the ability of pemphigus antibodies to fix complement in vitro is controversial. The purpose of this study was to test in vitro complement fixation abilities of pemphigus antibodies further using organ and tissue culture methods. Epidermal cell monolayers from mouse tail were incubated with the purified IgG fraction of pemphigus serum followed by purified Clq. Binding of Clq, as well as IgG was demonstrated by immunofluorescence methods. When purified Clq was replaced with normal human serum as a complement source, positive C3 and C4 staining were also evident. When purified IgG of normal human serum was used in place of pemphigus IgG, similar immunofluorescence staining was not observed. Further evidence for complement fixation in vitro by pemphigus antibodies was obtained using organ cultures. Organ culture of normal human skin and monkey esophageal mucosa cultured in purified pemphigus IgG showed intercellular substance binding of IgG. No binding was observed when normal IgG was substituted for pemphigus IgG. Additional organ culture sections were then treated with complement (fresh normal human serum) and tested by in vitro complement staining. Fixation of Clq, C4, and C3 was noted in intercellular substance areas of organ cultured skin and mucosa incubated with pemphigus IgG but not those incubated with normal IgG. Prior treatment of pemphigus IgG organ cultured skin sections with unlabeled anti-C3, blocked positive C3 staining. These results suggest that some pemphigus antibodies are capable of activating complement in vitro.

Animals↗
PubMed2005

Reversing the effects of formalin fixation with citraconic anhydride and heat: a universal antigen retrieval method.

Formalin is a commonly used fixative for tissue preservation in pathology laboratories. A major adverse effect of this fixative is the concealing of tissue antigens by protein cross-linking. To achieve a universal antigen retrieval method for immunohistochemistry under a constant condition, we developed a new method in which the effects of formalin fixation were reversed with citraconic anhydride (a reversible protein cross-linking agent) plus heating. Formalin-fixed, paraffin-embedded tissues from various organs were examined for immunohistochemical localization of a wide variety of antigens. Deparaffinized tissue sections were placed in an electric kitchen pot containing 0.05% citraconic anhydride solution, pH 7.4, and the pot was set at "keep warm" temperature mode of 98C for 45 min. This mode allowed heating the sections at a constant temperature. The sections were then washed in buffer solution and immunostained using a labeled streptavidin-biotin method using an automated stainer. In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult-to-detect antigens such as CD4, cyclin D1, granzyme beta, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions. This method provides efficient antigen retrieval for successful immunostaining of a wide variety of antigens under an optimized condition. It also allows standardization of immunohistochemistry for formalin-fixed tissues in pathology laboratories, eliminating inter-laboratory discrepancies in results for accurate clinical and research studies.

Antigens↗
PubMed2003

The microvasculature of the cerebral white matter: arteries of the subcortical white matter.

The microvascular architecture of the human cerebral subcortical white matter was studied. Most of the subcortical arteries ran straight through the cortex, but upon entering the white matter, they began to coil, loop, and spiral. Vascular stains showed wide spaces between the adventitial sheaths and blood vessels. The blood vessels coiled, looped, and spiraled within these wide adventitial spaces. This phenomenon was observed in the brains from persons ranging from the first to ninth decades of life and there were no statistically significant age-related correlations. Furthermore, there was no evidence of a reduction in the volume of white matter after fixation. Therefore, the observed tortuosity does not appear to be the result of shrinkage of brain tissue following fixation. While the mechanisms responsible for the subcortical arteries circuitry remain undetermined, this coiling architecture may serve as a trap for tumor cells and microorganisms passing through the blood stream, suggesting that these coiling arterial blood vessels may play a significant role in the pathogenesis of tumor metastasis and the brain abscess that frequently occurs in the gray-white matter junction.

Adolescent↗
PubMed1992

Immunohistological detection of human cytotoxic/suppressor T cells using antibodies to a CD8 peptide sequence.

AIMS: To evaluate whether cytotoxic/suppressor T cells can be detected in paraffin wax embedded human tissue samples using antibodies to a synthetic CD8 peptide sequence. METHODS: Polyclonal and monoclonal antibodies were raised against a 13 amino acid peptide sequence from the cytoplasmic portion of the alpha chain of the human CD8 molecule. RESULTS: These antibodies specifically detected the native form of the CD8 polypeptide when tested by immunoprecipitation with radiolabelled T cells, and gave the expected staining pattern for cytotoxic/suppressor T cells in cryostat sections. Being raised in rabbits, the polyclonal antibodies were also useful for double labelling for CD8 in conjunction with monoclonal antibodies. CD8 positive cells could also be detected in paraffin wax embedded tissues. This was achieved without prior treatment of the sections if the tissue had been fixed in Bouin's fixative. When tissues had been exposed to conventional formalin fixation, preliminary microwave treatment was required. CONCLUSIONS: These findings provide further evidence that antibodies against leucocyte associated antigens, capable of reacting on paraffin wax embedded tissue, can be produced by immunisation with synthetic peptide sequences.

Amino Acid Sequence↗
PubMed1980

A new one-step method for the cytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity.

A new one-step method for the light and electron microscopic localization of the ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na-K-ATPase complex is introduced. The incubation medium contains p-nitrophenylphosphate (NPP) as substrate, lead citrate as the capture reagent, and dimethylsulfoxide (DMSO) as an activator. It is usable at the optimal pH of the K-NPPase, which is about pH 9.0 in the presence of 25% of DMSO. The effects of fixation, lead concentration, and DMSO on the enzyme activity were studied using rat kidney as a test tissue. The fixation of tissues in a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde for 60 min at 0 degrees--4 degrees C preserved 45% of the enzyme activity. In the absence of DMSO, lead citrate (4.0 mM) caused 82% inhibition of the enzyme activity in fixed tissue. However, the addition of DMSO (25%) caused about 3-fold activation of the remaining activity. Cytochemical demonstration of the ouabain-sensitive K-NPPase activity was successfully made by this method at both light and electron microscopic levels.

4-Nitrophenylphosphatase↗
PubMed1992

Immunocytochemistry of neurofibrillary tangles with antibodies to subregions of tau protein: identification of hidden and cleaved tau epitopes and a new phosphorylation site.

Antibodies to multiple epitopes spanning the length of the tau molecule were used to study Alzheimer neurofibrillary tangles (NFT) using immunocytochemical methods and several different methods of fixation and tissue processing, including staining of vibratome sections, hydrated autoclaving of paraffin sections and immunofluorescence of NFT isolated from fresh brain tissue. Smears and sections were pretreated with trypsin and/or phosphatase to further characterize antibody binding. In tissue fixed briefly in periodate-lysine-paraformaldehyde, tau immunoreactivity was detected in astrocytes, but only a few tau epitopes were detected in NFT with this fixation method. In contrast, all tau epitopes were detected in NFT in tissue fixed in formaldehyde for prolonged periods of time. In the hippocampus, the number of NFT detected in the dentate fascia was in proportion to the duration of dementia, as we previously noted. Dentate fascia NFT were intracellular (i-NFT) and were reactive with antibodies recognizing epitopes in both the carboxy- and amino-terminal regions of tau, but not the microtubule-binding domain of tau, suggesting that microtubule-binding domain epitopes are hidden in i-NFT. In contrast, NFT in the subiculum and layer II of the parahippocampal cortex were mostly extracellular (e-NFT), especially in severe cases of long duration. e-NFT were immunoreactive with antibodies to the microtubule-binding domain, but only weakly reactive with antibodies to carboxy- or amino-terminal epitopes, suggesting that e-NFT may contain fragments of tau. In both isolated NFT and NFT in sections, amino-terminal epitopes, including the Alz-50 epitope, were sensitive to trypsin proteolysis, which suggests that the lack of staining of e-NFT by antibodies to the amino-terminal regions of tau is due to proteolysis. Antibodies reactive with amino-terminal epitopes also stained fewer NFT following hydrated autoclaving, while those reacting with the carboxy half of tau stained more NFT after hydrated autoclaving. Thus, although carboxy-terminal regions are not detected in e-NFT, they are probably masked, rather than proteolytically cleaved, since they can be revealed by hydrated autoclaving. Finally, phosphatase treatment of isolated NFT revealed enhanced immunostaining not only with Tau-1, as in previous studies demonstrating abnormal phosphorylation of tau proteins in NFT, but also with an antibody to exon 2, which reveals yet another phosphorylation site in tau of NFT.

Aged↗
PubMed2005

Transpiration-assisted perfusion fixation provides in situ preservation of developing ray parenchyma cells in Eucalyptus nitens.

The technique of animal vascular perfusion fixation was adapted for in situ fixation of the fragile and difficult to access cells of the ray parenchyma system in stems of 10-year-old Eucalyptus nitens trees. In situ fixation enabled tissue to be safely dissected for histological processing without risk of damage to microstructure or initiation of wound response. Fixative was perfused through the active vascular system under hydrostatic and transpiration pressure directly to vessel-associated ray cells. Diffusion from vessels allowed fixative to access nonvessel-associated ray cells. Acrolein was included to aid diffusion fixation and safranin dye was included to define fixed regions within the xylem. Sections prepared for light and electron microscopy from samples cut from regions showing intense safranin staining showed good microstructural preservation and were free of artefacts caused by mechanical injury or wound response.

Eucalyptus↗