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PubMed1999

Immunocytochemistry of lipids: chemical fixatives have dramatic effects on the preservation of tissue lipids.

We report here the effects of chemical fixatives on lipids studied under conditions simulating the immunogold labelling of phosphatidylserine. Using anti-phosphatidylserine antibodies, it is shown that the labelling intensity of a phosphatidylserine/phosphatidylcholine coating depends largely on the conditions of fixation. In fact, the usual aldehydic fixatives washed out most of the phostphatidylserine, thus preventing the binding of anti-phosphatidylserine antibodies. This was confirmed on biological samples such as rat liver and brain by measuring the loss of radiolabelled lipids during the fixation procedure. Furthermore, the complete procedure of tissue preparation for electron microscopical observation was investigated. The loss of (radiolabelled) lipids was studied in tissue samples during fixation and resin embedding. The results showed that the classical procedure (glutaraldehyde fixation followed by epoxy resin embedding) results in the loss of 73-91% of the tissue lipids whereas in unfixed, freeze-substituted samples, more than 76% of the tissue lipids are preserved.

Animals↗
PubMed1989

Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences.

Conditions for combination of DNA in situ hybridization, using biotinylated DNA probes, with immunohistochemistry were investigated on cryostat sections, cytological preparations, and paraffin sections. We found that cryostat sections and cytological preparations are suitable for in situ hybridization of target DNA after fixation in acetone, methanol, ethanol, or Carnoy without further proteinase pretreatment. Acetone is also very suitable for immunostaining of cell surface or cytoskeleton antigens. We therefore performed combined immunoenzyme and in situ hybridization staining using this fixative. The best results were obtained when immunoperoxidase staining with diaminobenzidine/H2O2 was followed directly by in situ hybridization. In addition to immunoperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) staining with naphthol ASBI phosphate and New Fuchsin as a substrate could be used. In most instances, detection of the biotinylated hybrid with a streptavidin-biotinylated polyalkaline phosphatase method using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as the substrate was preferable. The double stainings were studied on the following test models: (a) frozen tonsil sections: cell surface antigens (pan T) and ribosomal DNA; (b) frozen genital condyloma sections; cytokeratins and human papillomavirus type 6 + 11 (HPV-6/11) DNA; (c) CaSKi cells: cytokeratins and HPV-16 DNA; (d) infected fetal lung fibroblasts: vimentin and cytomegalovirus (CMV) DNA. An adapted procedure was followed on routinely formaldehye-fixed and paraffin-embedded condyloma tissue. Immunoperoxidase staining for papilloma virus capsid antigen could be combined with DNA in situ hybridization with HPV-6/11 DNA. In this model, however, the accessibility of the target DNA had to be improved by enzyme treatment after the immunostaining and before starting the in situ hybridization.

Antigens, Surface↗
PubMed1988

Interspecies comparisons of tissue DNA damage, repair, fixation, and replication.

The many anatomical, physiological, and biochemical differences among various mammalian species make it difficult to extrapolate carcinogenic potency data from animals to humans. The process is further complicated by the multistep origin of most malignant tumors in animals and humans due to the interaction of target cells with both endogenous and exogenous factors. Species differences in these aspects of carcinogenesis must also be considered when attempting to evaluate the carcinogenic risks of chemicals to humans. Cancer development in animals involves at least three distinct stages: initiation, promotion, and progression. Intra- and interspecies differences in susceptibility to carcinogenesis may be related to any one or a combination of these stages. Variation in species susceptibility to tumor initiation may result from differences in the abilities of various species to metabolize a potential carcinogen to an ultimate carcinogenic form and/or to detoxify the carcinogen. Most comparative studies among species have only revealed subtle differences in metabolism. DNA adducts from several activated carcinogens have been found to be the same in a number of tissues from various species, including humans. Capacity for DNA repair is apparently a critical factor in the initiation of carcinogenesis in target cells of different species but is less critical among mice that differ in susceptibility to two-stage carcinogenesis of the skin and liver. Susceptibility variations among stocks and strains to such carcinogenesis appear to be related to alterations in tumor promotion. Additional comparative studies are critically needed on all aspects of carcinogenesis to permit effective extrapolation of carcinogenic potency data from animals to humans.

Animals↗
PubMed2006

Latest developments in in situ PCR.

Since the first publication on the method of in situ polymerase chain reaction (PCR), several thousand research papers have appeared in peer-reviewed journals describing various findings based solely on the application of this method or combined with other more robust methods, including solution-based PCR, immunohistochemistry, Southern blot, etc. A few years after the advent of PCR, several investigators developed in situ PCR methods that differed considerably from each other with regard to tissue preparations, fixation, mounting of slides, reverse transcription technique, primer design, target selection, size, and amplicon size, and thermocycler designs and the use, among many other fine details. This chapter describes the detail procedures that are used in the author's laboratory. It also discusses the variations and modification that can be used for the specific needs of an investigator. This protocol should serve as a primer for the investigators, and each researcher must use his or her variation according to their needs.

Cells, Cultured↗
PubMed2001

Unmasking antigens for immunohistochemistry.

Preservation of tissue by fixation in a formalin solution, followed by dehydration and paraffin-wax embedding remains the predominant method of preparation for microscopic analysis of morphology. Whilst this may be optimal for morphological assessment, this technique has major disadvantages for subsequent immunohistochemical study as a result of the structural alteration of antigens that occurs during the processing procedure. However, the introduction of a range of antigen unmasking procedures has revolutionised immunohistochemistry, establishing the technique as a powerful tool in diagnostic pathology. Here, the development of enzymatic, non-enzymatic and heat-based antigen retrieval techniques is reviewed, and the methods in use currently are appraised.

Antigens↗
PubMed1994

Immunohistochemical localisation of glucose-6-phosphatase in developing human kidney.

The objective of our study was to determine the cellular localisation of glucose-6-phosphatase in developing human kidney using monospecific antiserum and a standard immunohistochemical method (peroxidase-antiperoxidase, PAP) on formalin fixed and paraffin embedded tissue. In embryonic and early fetal development of the metanephric kidney, glucose-6-phosphatase is located primarily in derivatives of the ureteric bud such as the pelvis, calyces and collecting ducts. In mid-fetal life as nephrons evolve and develop they become increasingly immunoreactive to glucose-6-phosphatase, such that in mature metanephric kidney the proximal tubules are highly reactive for glucose-6-phosphatase with other elements of the nephron also immunopositive albeit at lower reactivities. In addition the parietal layer of Bowman's capsule and some cells of the visceral layer are immunopositive. Only with the development of nephrons does the early predominance of glucose-6-phosphatase immunoreactivity to ureteric bud derivatives change: in mature kidney the reactivity in the collecting ducts is a small proportion of the total. In proximal tubular cells the distribution of glucose-6-phosphatase immunoreactivity is relatively uniform throughout development in contrast to collecting ducts where in fetal life this reactivity is displaced to the apices and basal areas by intracellular glycogen deposits. The mesonephric kidney has a similar pattern of glucose-6-phosphatase immunoreactivity to that of metanephric kidney. The availability of monospecific antiserum to glucose-6-phosphatase and immunohistochemical methods now allows an alternative approach to cellular localisation. Many of the difficulties in the fixation of tissue and assay of glucose-6-phosphatase activity inherent in conventional histochemical methods are avoided by such methods.

Female↗
PubMed2004

Staged management of high-energy proximal tibia fractures.

High-energy proximal tibia fractures are complicated by soft tissue compromise and this may result in sub-optimal outcomes. There is a high association of open injuries, compartment syndromes, and vascular injuries with these bony disruptions. Surgical treatment of these injuries has been associated with significant complications such as infection, knee stiffness, malunion, loss of fixation, soft tissue failure, and amputations. The loss of fixation is an issue especially in the elderly, with failure associated with age more than sixty years, premature weight bearing, preoperative displacement, fracture fragmentation, and severe osteoporosis. The use of two-stage reconstruction for the treatment of distal tibia fractures has been successful in decreasing the complication rates, including wound compromise. The two stages involve: 1. stabilization of the injured limb with a bridging external fixator to allow the soft tissues to improve and recover and 2. definitive fixation for reconstruction of the articular surface and meta-diaphyseal fractures. The use of such a protocol has been proposed for high-energy proximal tibia fractures to decrease the high rate of soft tissue compromise associated with traditional open methods of treatment. The choice of definitive fixation may include plates, nails, or non-bridging external fixation.

Adult↗
PubMed1999

Survival-fixation of the cochlea: a technique for following time-dependent degeneration and repair in noise-exposed chinchillas.

To minimize problems with data interpretation due to interanimal variation in susceptibility to noise, we developed a survival-fixation paradigm which involves fixing one cochlea of an experimental chinchilla at one post-exposure time and fixing the second cochlea as much as 14-24 days later. This paradigm is analytically effective because there is a high correlation in the magnitude and pattern of damage in the left and right cochleas of binaurally exposed animals. Thus, each experimental animal provides two snapshots in the degeneration and repair continua in which it can be certain that both cochleas sustained equivalent amounts of damage during the exposure. Using this technique, the time course of degeneration of different structures and cells in the organ of Corti can be determined and primary damage can be distinguished from secondary effects. The present paper discusses the issues which had to be addressed to develop this technique and provides preliminary results from chinchillas exposed to a traumatic noise.

Animals↗
PubMed1981

Cytofluorometry on cells isolated from paraffin sections after blocking of the background fluoroscence by azocarmine G.

A technique for isolation of cells from paraffin embedded tissue is indispensable for the performance of Feulgen-DNA cytofluorometry in parallel with the definition of histological characteristics. Background fluorescence due to nonspecific dye-binding by a "pseudo-plasmal reaction" is usually found to be so intense on cells isolated from formalin-fixed tissues, that we are often forced to abandon quantitative DNA determinations. In the present work, we report the fixation of tissues with Carnoy's fixative for 12 h at 5 degrees C not only reduces nonspecific dye-binding but also facilitates the process of cell isolation. Furthermore, we find that pre-treatment of cells isolated from Carnoy-fixed tissues with acidic azocarmin G solution completely blocks nonspecific dye-binding in subsequent acriflavine Feulgen nuclear staining. This combination of techniques for specimen preparation enables us to carry out Feulgen-DNA cytofluorometry on cells isolated from histological sections with satisfactorily low coefficients of variation (less than 8%). The techniques should be widely applicable for parallel DNA determinations and histology.

Animals↗
PubMed1975

The effects of various fixative-buffer combinations on lung fine structure.

A variety of fixatives, buffers and fixation procedures were compared in rat and squirrel monkey lung in an attempt to preserve optimally both the cytologic details of pulmonary parenchyma as well as the acellular avelolar lining layer. In initial experiments utilizing the fixative of Ito and Karnovsky ('68), an electron-dense deposit was observed on the alveolar surface. Experiments were carried out in an attempt to determine what component of this fixative was responsible for the reaction product observed. In addition,immersion fixation of tissue blocks was compared to the whole lung fixation method of Kikkawa ('70). Kikkawa ('70) achieved excellent preservation of the acellular alveolar lining layer by such a fixation technique. In all lungs examined, whenever a phosphate buffer was utilized with primary aldehyde fixation, an electron-dense precipitate was observed on the luminal surfaces of the type I and II pulmonary epithelial cells. Additional sites of reaction product were pinocytotic vesicles of the type I cells and membranous arrays within the alveolar lumen. Such deposits were never observed when a sodium cacodylate buffer was used. No such granules were observed in areas of lung where the acellular alveolar lining layer had been preserved. The implications of these findings with regard to lung histochemical procedures and the possible relationship of these phosphate buffer-dependent granules to the surfactant system are discussed.

Animals↗
PubMed1981

The cytochemical demonstration of oestrogen receptors in human breast carcinomas.

A cytochemical method has been used to demonstrate oestrogen receptor in histological sections of breast carcinomas, and has given similar qualitative results to the biochemical dextra-coated charcoal method. Two conjugation techniques were employed to couple horseradish peroxidase to 17 beta-oestradiol-6-O-carboxymethyloxime-bovine serum albumin. The periodate conjugation method led to a greater degree of coupling but a higher concentration was required for demonstration of oestrogen receptor than that needed when the two-stage glutaraldehyde conjugation method was use, even though this resulted in a lesser degree of coupling. Care in the preparation of tissue has been found to be essential. Formalin-fixed, paraffin-embedded tissue was unsuitable; rapid freezing of fresh tissue, only brief air-drying or acetone fixation of tissue sections and short-term storage were important. A correlation has been shown between the presence of oestrogen receptor in breast carcinomas and good histological differentiation. The poorer differentiated tumours were also found to contain fewer reactive cells.

Breast Neoplasms↗
PubMed1997

Compression plating of tibial fractures following primary external fixation.

During the time period from May 1990 to December 1992, a total of 75 tibia fractures were treated in the Department of Traumatology at the University of Bonn. Thirty-eight patients with 40 tibial fractures were managed according to a regimen including primary stabilization, usually using external fixation, soft tissue reconstruction and delayed open reduction and internal fixation using an AO compression plate. The majority of the patients had been involved in motor vehicle accidents, leading to multiple injuries in 24 instances. An open fracture was seen 18 times. The 20% complication rate is comparable to the reports following intramedullary stabilization. Only one infection, following a grade 2 open fracture, was seen after the definitive stabilization. Bony union was achieved after 15.7 weeks. In light of the complications associated with intramedullary nailing, such as fat or air embolism, heterotopic ossification and non- or malunions, use of the tibial plate does not offer just logistic advantages, but is a viable alternative for delayed stabilization of tibial fractures. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. No funds were received in support of this study.

Adult↗
PubMed1975

[Internal fixation with soft-tissue lesions: fracture with nerve damage (author's transl)].

Typical nerve damage after fractures due to the topographical proximity of the nerves to the bones, are shown and therapeutic principles are discussed. Primary traumatic lesions are differentiated from so-called "creeping paralysis". The methods of choice are neurolyses, nerve suture or transplantation, each at the appropriate time. Evaluation of 83 of the 128 Patients undergoing microsurgical operations on the nerves in recent years is presented after re-exmination by independent neurologists. One-third showed definite signs of regeneration, more than one-third had good functional results, and poor results were recarded for 25 percent of the patients.

Fractures, Bone↗