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PubMedDate not supplied

Immunohistochemical investigation of the human inner ear. Limitations and prospects.

The application of highly sensitive and specific immunohistochemical methods in routine light microscopy has been rewarded by a great many new observations. The essential underlying antigen-antibody reaction has allowed the recognition and identification of some unknown or unidentified components of the cell. Successful results are best achieved by means of reliable fixation and the use of frozen sections. Frozen sections of the human cochlea are, however, impaired by unavoidable damage to the tissues to be studied. The temporal bone has to be decalcified, resulting in a reduction of the range of suitable methods and an interference with a reliable interpretation of the results. The preservation of the specific antigenicity of the tissues is of paramount importance, allowing the detection of the investigated antigen by the antibody applied. The investigation of different fixatives and various methods of decalcification in tissues possessing some familiar and readily identifiable antigens has confirmed the immunohistochemical suitability of properly fixed and adequately decalcified human temporal bones. The present demonstration deals with some of the principal technical procedures and includes examples of their application for the study of the human inner ear. The goal of our research is the development of reliable immunohistochemical methods of fixation and decalcification to be employed in the study of specimens from patients with Ménière's disease, sudden deafness, progressive loss of hearing, genetic sensorineural syndromes, etc. This will lead to the extension of our knowledge of some unrecognized causes of sensorineural hearing loss.

Ear, Inner↗
PubMed1995

[The new Hannover method of synthetic embedding of bone marrow. Cold polymerization of methylmethacrylate].

A patented low-temperature polymerization method for methylmethacrylate (MMA) infiltrated bone marrow biopsies is described: it has been developed from our previous MMA technique and is a patented procedure. Differences from the previous method are (1) removal of stabilizer from the MMA monomer before its application, (2) the use of a different starter, (3) avoidance of O2 influence during polymerization by means of vacuum exchange with N2, and (4) polymerisation in a water bath to draw off residual heat. After this procedure, all immunohistochemical reactions are possible provided that the previous fixation is adequate. The effects of different fixatives are reviewed briefly without detailed analysis. Technically, this plastic embedding can be performed at least as rapidly as the classic paraffin embedding after decalcification. The advantages over the latter method are: (1) the cells can be better differentiated because semi-thin sections can be made; (2) the immunoreactions can also be performed on the basis of semithin sections, which means they can be interpreted more easily; (3) morphometric analyses yield more reliable results because of the constant thickness of sections; (4) osteological examination of bone trabeculae, especially the search for mineralisation deficiencies, is possible; (5) the plastic embedding procedure is less dependent on individual instabilities in the quality of performance of the staff members involved. Furthermore, it is worth mentioning that the costs for additional equipment necessary remain below DM 100,000 including an excellent microtome.

Biomarkers, Tumor↗
PubMed1983

A new technique for the study of temporal bone pathology.

A new technique for the study of the temporal bone at autopsy is presented in which thin slices of the fixed bone are prepared on a special cutting machine which preserves the configuration of the delicate anatomical structures. Selected areas are then processed for light microscopy. There is thus opportunity for gross examination, only a short period for immersion in acid for decalcification if required, and special histological procedures may also be carried out. The method is quick and technically easier than serial sectioning and storage of the smaller numbers of sections from each case is less of a problem. Temporal bones which have been perfused through the perilymph within 3 h of death may also be cut in the same way in order to allow removal of samples of membranous labyrinth for surface preparation or electron microscopy, avoiding the need for drilling to reach the membranous labyrinth, and being quicker and less damaging.

Histological Techniques↗
PubMed1992

Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections.

The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all active parts of the cell cycle. Recently we have raised monoclonal antibodies, MIB 1-3, against recombinant parts of the Ki-67 antigen. These antibodies are true Ki-67 equivalents, as demonstrated by immunostaining of fresh specimens, biochemistry, and molecular biological techniques. Formalin-fixed, paraffin-embedded sections routinely processed for immunohistochemistry failed to stain for Ki-67 and MIB 2. Antibodies MIB 1 and MIB 3 labelled mitotic figures, while non-mitotic proliferating cells were negative under these conditions. However, when dewaxed microwave oven-processed paraffin sections of formalin-fixed tissues were used, MIB 1 and MIB 3 gave strong nuclear staining of those cells presumed to proliferate under a variety of normal and neoplastic conditions. Moreover, routine decalcification or depigmentation techniques did not alter the immunoreactivity of MIB 1 and MIB 3 with microwave-processed paraffin sections. This method is highly reproducible, easy to perform at low cost, and no additional technical skill is needed because after microwave treatment just routine immunohistochemical methods are used. Since we have successfully applied this new method to sections obtained from paraffin blocks stored for a long time (in one case more than 60 years), the assessment of cell kinetics through the detection of Ki-67 antigen is now possible on archival material collected in histopathology departments all over the world.

Antibodies, Monoclonal↗
PubMed2001

Vascularity of the carpus.

Relatively few studies investigating the vascular patterns of the carpus have been performed. Technical difficulties in identifying small vessels in three dimensions and in determining their location within the thick ligaments about the wrist have led to conflicting anatomic reports. Studies on cadavers in which improved techniques with arterial injection, chemical debridement, and decalcification were used allowed the arterial anatomy of the carpus to be delineated more accurately. The current authors review these arterial patterns, with attention given to the extraosseous and intraosseous vascularities.

Carpal Bones↗
PubMed1999

Improved RNA analysis for immediate autopsy of temporal bone soft tissues.

RNA analysis is essential for understanding biological activities of a cell or tissue. Unfortunately, retrieval of RNA from existing archives of human temporal bones has proven extremely difficult due to degradation of RNA molecules. The major factors that contribute to degradation of RNA in specimens from autopsied temporal bones are tissue autolysis due to time elapsed before autopsy, and technical problems in processing the bones after harvest. We therefore focused on improving the survival of RNA in human temporal bones by shortening the time to autopsy and through modification of the processing technique by removing targeted tissues directly from the temporal bones and by avoiding time-consuming decalcification and celloidin-embedding. Eight temporal bones collected at immediate autopsies were used in this study. Representative mRNAs, ranging from high (MUC5B, physically unstable) to low (beta-actin, physically stable) molecular weights, and from abundant (MUC5B) to non-abundant (MUC1) RNA, were studied by in situ hybridization, Northern blot technique, or both. Using this modified protocol in autopsies performed up to 6 h after death, the existence of mRNAs was demonstrated in all bones studied. This improved method demonstrates the feasibility of the use of autopsied temporal bone tissues for RNA analysis.

Autopsy↗
PubMedDate not supplied

Horizontal splitting in patellar articular cartilage.

Horizontal splits at the interface between the uncalcified and the calcified layers of the articular cartilage have been found in 31 of 50 transverse slabs of left patellae from a random series of 50 necropsy subjects aged 18 to 96 years, Evidence is presented that the splits are not due to autolysis nor to technical artefact, and it is here suggested that they result from shearing damage at the uncalcified-calcified cartilage interface during life.

Adolescent↗
PubMed1997

Plastination of the larynx for whole-organ sectioning.

Whole-organ sectioning is an important technique for the assessment of laryngeal pathology. Since currently established methods require prior decalcification which causes morphological changes, the critical border area between cartilage and surrounding soft tissue cannot be investigated in the same specimen and morphometric studies are not possible. Plastination is a laboratory technique that has previously demonstrated its capacity to overcome these shortcomings. In so doing water and lipids are replaced by curable polymer within the laryngeal cells making decalcification unnecessary. In the present study, more than 50 human larynges were processed using block plastination (BP) and sheet plastination (SP). For BP the complete organ was plastinated as a whole and then cut into thin serial sections. For SP the fresh organ was sliced first and plastinated in a second step. Findings demonstrated that SP allowed for the production of whole-organ sections within a period of 1 week only. Section thicknesses were as thin as 15 mm using a diamond wire saw and an ultramilling device. Sectioning was possible in both coronary and horizontal planes. Following BP, specimens were cut in an industrial cutting machine to thicknesses of about 0.6 mm. Shrinkage of tissue was less than 10% for both methods. In all, SP was technically superior to routine paraffin histology, although cutting equipment is very expensive and delicate in handling. At present the technique of BP is the method of choice for macromorphometrical investigations on serial sections of the human larynx.

Acetone↗
PubMed1981

[Man in the condition of weightlessness (author's transl)].

In the space flights by the USA and USSR to date adequate experience has been obtained on the adaptation of man to living conditions in space--with the technical assistance of an artificial protective atmosphere in the space ship or space suit--and to the singular factor of "weightlessness" (microgravity, low gravity) and how he re-adjusts again after returning to a gravitational field. There is no ultimate or absolute physiological barrier for long term space flight--with one exception: decalcification of the skeleton. For short space flights, however, space sickness is a serious disorder.

Adaptation, Physiological↗
PubMed1989

Bone core biopsies. Plastic or paraffin?

Plastic embedding of bone core biopsy specimens has been promoted as providing superior morphology, primarily because semi-thin sections can thereby be cut at 1-2 mu. The major disadvantages of plastic embedding are that it increases the technical load, is more expensive, and potentially has its own intrinsic problems, including difficulties in performing special stains and immunoperoxidase studies. In order to investigate the possibility that semi-thin paraffin sections may provide similar morphological results without the intrinsic disadvantages of plastic sections, we examined 45 bone core biopsy specimens that were sufficiently large to process one half in plastic and the other half in paraffin following decalcification. Both were cut at 1-2 mu. Although many plastic sections appear esthetically more pleasing, semi-thin paraffin sections of very high quality can also be obtained routinely. Additional advantages of paraffin sections were the ability to perform peroxidase studies, lower cost, less technologist time, and avoidance of problems occasionally arising with plastic, such as difficulties with impregnation or problems with polymerization. Peroxidase studies were particularly useful in patients with possible myeloma that was not overt on hematoxylin-and-eosin section and in confirming the presence or source of metastatic carcinoma. We therefore recommend the use of semi-thin (1-2 mu) paraffin sections for routine examination of bone core biopsy specimens.

Biopsy↗
PubMed2002

Fixation and decalcification of adult zebrafish for histological, immunocytochemical, and genotypic analysis.

To facilitate the molecular analysis of tissues in adult zebrafish, we tested eight different fixation and decalcification conditions for the ability to yield DNA suitable for PCR and tissue immunoreactivity, following paraffin embedding and sectioning. Although all conditions resulted in good tissue histology and immunocytochemistry, only two conditions left the DNA intact as seen by PCR. The results indicate that zebrafish fixed in either 10% neutral buffered formalin or 4% paraformaldehyde, followed by decalcification in 0.5 M EDTA, is an easy and reliable method that allows molecular experiments and histology to be performed on the same specimen. The fixation and decalcification by Dietrich's solution permitted the PCR amplification of DNA fragments of 250 but not 1000 bp. Therefore, a protocol of formalin or paraformaldehyde fixation followed by decalcification with EDTA is broadly applicable to a variety of vertebrate tissues when excellent histological, immunocytochemical, and genotypic analyses may be simultaneously required.

Animals↗