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PubMed2003

Immunohistochemical identification of multipotent adult progenitor cells from human bone marrow after transplantation into the rat brain.

In the immunohistochemical analysis of the brain, tissue preparation and fixation are critical steps. During our studies of transplanting human bone marrow multipotent adult progenitor cells (hMAPCs) into the rat brain, we noticed that various methods of brain tissue preparation and fixation differentially influenced antigenic preservation in the transplants and in the host brain. Here, we report a simple, effective and reproducible method of tissue preparation and fixation that results in the immunohistochemical labeling of transplanted and host cells.

Animals↗
PubMed1992

Variability of laminin immunoreactivity in human autopsy brain.

Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.

Adult↗
PubMed1993

Ultrasonic-pathological comparison of the human arterial wall. Verification of intima-media thickness.

Recent intravascular ultrasound experience challenges the accuracy of ultrasonic measurement of arterial wall thickness. We reevaluated the correlation between histological and sonographic measurements of intima-media thickness using standard transcutaneous vascular technology. Carotid and femoral arterial segments were imaged before and after fixation using a 7-MHz linear-array vascular transducer. Log compression and beam orientation were varied. Mean intima, media, and adventitia thicknesses were measured and compared with corresponding histological tunica. Tissue processing caused 2.5% shrinkage. Intraobserver reading error was 0.7% for histology and 5.4% for sonography. Ultrasound overestimated the thickness of the intima and adventitia and underestimated the thickness of the media. For combined intima-media thickness, the differences between histology and imaging were insignificant, averaging 4% for the carotid artery and 9% for the femoral artery in the far-wall projection. In the near-wall projection, sonographic intima-media thickness was 20% less than that determined histologically. We conclude that ultrasonography is limited mainly by axial resolution in quantifying the dimensions of individual arterial tunica but is capable of accurately measuring far-wall intima-media thickness.

Aged↗
PubMed1989

Correlation of c-myc expression with nuclear pleomorphism in human renal cell carcinoma.

The expression of the c-myc gene product in renal cell carcinomas was examined by immunostaining with monoclonal antibody (mAb) MYC-1. The effects of preservation and fixation of tissues on staining were first examined. In cryostat sections fixed with 4% buffered formalin for 15 min, staining was observed in the nucleus. On the other hand, in paraffin sections after fixation with 10% formalin, staining was observed in the cytoplasm, but not in the nucleus. Because c-myc protein has been shown to be a nuclear protein, the finding that c-myc protein was not detectable in the nucleus appeared to be due to the preservation or fixation procedures used. Therefore, cryostat sections fixed with 4% formalin were used to investigate the correlation between the reaction of MYC-1 mAb and nuclear pleomorphism in primary and metastatic renal cell carcinomas. Among 41 primary tumors, positive staining was observed in 2 of 17 tumors (12%) of grade 1, 17 of 21 (81%) of grade 2, and all 3 (100%) of grade 3. Among 17 metastatic tumors, positive staining was not observed in any of the 5 (0%) of grade 1 but was observed in 2 of 4 (50%) of grade 2 and all 8 (100%) of grade 3. Thus, the frequency of the positive reaction with MYC-1 mAb was correlated with nuclear pleomorphism in primary and metastatic renal cell carcinomas. The reaction of Ki-67 mAb, which recognized a nuclear antigen present in proliferating cells, was also correlated with nuclear pleomorphism. These findings suggest that the c-myc gene product plays a role in cell proliferation in renal cell carcinomas.

Antibodies, Monoclonal↗
PubMed1975

[Method of determination of fat deposit in chickens].

The method described is based on the histochemical demonstration of fats with the use of Sudan stains and the clearing of tissues in an alkaline medium. It is employed to investigate whole birds, thick sections or individual organs to determine the morphotopographic localization of the fat depots. The procedure comprises the alcohol fixation of tissues, the staining of lipids with Sudan black, the clearing of tissues in a solution of potassium hydroxide, and the final mounting of the cleared materials in glycerine. This treatment results in that the sites of fat deposition assume a blue coloration, the bones are grey, and all remaining tissues and organs (if properly bled) are colorless and transparent. The method can also be successfully used with materials that have been preliminary fixed with formalin but their clearing has been considerably worse.

Animals↗
PubMed2002

LR White is preferable to Unicryl for immunogold detection of fixation-sensitive nuclear antigens.

The purpose of this study was to compare two electron microscopy embedding media - LR White and Unicryl - with regard to cell morphologyical and immunohistochemical preservation properties for the study of fixation-sensitive nuclear antigens. Human cervical carcinoma (HeLa) cells were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde, and embedded in parallel in the two resins: LR White and Unicryl using; two different polymerization protocols were used for each resin. Preservation of fine nuclear structure was good after LR White and poor after Unicryl embedding. Immunogold labeling of Sm antigen was significantly stronger on LR White sections. Polymerization by UV light resulted in stronger and more specific labeling than heat polymerization. These results show that LR White is advantageous over Unicryl for the study of nuclear antigens requiring delicate aldehyde fixation.

Acrylic Resins↗
PubMed1978

Techniques of bone fixation and clinical experience in replanted extremities.

Bone is shortened only enough to obtain skin closure or to remove excessive local comminution. Shortening is never done solely for the purpose of obtaining vessel apposition since vascular damage may extend beyond the zone of general soft tissue involvement. Fixation should be rapidly accomplished and be reasonably stable. Techniques are described. Following bony fixation, repair of flexor tendons and then nerves is carried out. Vessel repair using vein grafts to make up defects is then accomplished followed by loose skin closure.

Adolescent↗
PubMed1997

Identification of leucocyte surface antigens in paraffin-embedded bovine tissues using a modified formalin dichromate fixation.

A modified fixative of formalin dichromate was combined with a cold embedding procedure for the preservation of bovine leucocyte surface antigens. Fourteen monoclonal antibodies recognizing seven bovine leucocyte surface antigens (BoCD1w2, BoCD4, BoCD8, BoWC1, BoWC3, BoWC4 and BoIgM) were applied as primary antisera in a sensitive avidin-biotin-peroxidase complex detection method. The staining results were compared with those obtained in cryostat and routinely formalin-fixed sections of corresponding tissue samples. Using the modified formalin dichromate fixative and the cold embedding procedure, all the leucocyte surface antigens tested were detectable immunohistologically in paraffin sections with a generally more distinct staining than in traditionally processed tissues. Morphological structures were better preserved than in cryostat sections but, to some extent, were poorer when compared with routinely formalinfixed tissues. However, this method suggests that there are only mild masking effects and provides an alternative to the use of unfixed material, particularly for morphological-immunohistochemical investigations.

Animals↗
PubMed2000

Tyrosinase immunoreactivity in fine-needle aspiration samples of metastatic malignant melanoma.

BACKGROUND: Tyrosinase, the rate-limiting enzyme in melanin synthesis, is a melanoma associated antigen that is recognized by both CD4+ and CD8+ T-cells in an HLA-restricted fashion. Peptides derived from the tyrosinase antigen currently are being utilized as a target for T-cells in several immunotherapy protocols for metastatic malignant melanoma (MMM) at the National Institutes of Health/National Cancer Institute. Serial fine-needle aspirations of metastatic lesions are performed to monitor the antigen expression of tyrosinase during treatment by immunostaining cytologic preparations with the monoclonal antibody T311. METHODS: In the current study, 62 samples of MMM were evaluated for tyrosinase immunoreactivity on air-dried, acetone fixed cytospins and the corresponding formalin fixed, paraffin embedded cell block using an avidin-biotin immunoperoxidase method. RESULTS: Positive immunoreactivity revealed a granular cytoplasmic staining in melanocytic cells. The current study results showed that 92% of samples (57 of 62) were T311 immunoreactive on cell block preparations, whereas only 61% (38 of 62) were immunoreactive on cytospin preparations. In 66% of samples (41 of 62) immunoreactivity for T311 was greater in the cell block sample than in the corresponding cytospin, whereas in only 3% of samples (2 of 62) was it greater in the cytospins. In 31% of samples (19 of 62) there was no significant difference in immunoreactivity between the 2 sample types. CONCLUSIONS: The results of the current study show that tyrosinase is a sensitive marker for the detection of MMM; however, the optimal method of sample preparation for immunoperoxidase staining appears to be formalin fixation and paraffin embedding as tyrosinase immunoreactivity is diminished significantly in air-dried cytospin samples despite subsequent acetone fixation. Cancer (Cancer Cytopathol)

Acetone↗
PubMed1992

Characterization of inner ear sensory hair cells after rapid-freezing and freeze-substitution.

In order to determine the ultrastructural organization of normal cells and to understand better the anatomical substrates for outer hair cell motility, cryofixation was performed on the sensory epithelium of the inner ear of guinea pigs prior to substitution of frozen water with organic solvents containing chemical fixatives. In this way cells would not be altered by the direct application of the chemicals commonly used for preservation, which are also known to cause fixation-induced shape changes in outer hair cells. Following rapid freezing and freeze-substitution, preservation of cells within the isolated sensory epithelium containing the organ of Corti was similar to that seen in conventionally fixed cells. However, in rapidly frozen and freeze-substituted outer hair cells the cytoplasm and the cellular membranes differed from those seen in conventionally fixed preparations. The cytoplasmic matrix was densely packed with filaments and stained homogeneously, suggesting better preservation of the cytoskeleton and less extraction of the soluble ground substance. Cell membranes were smooth, indicating that fixation-induced shape changes and shrinkage had been avoided. The subsurface cisternal system of intracellular membranes lining the lateral wall of the outer hair cells was composed of continuous, tightly packed, parallel rows of membranous lamellae. Thus rapid-freezing and freeze-substitution are important techniques by which structural alterations correlated with outer hair cell motility can be separated from fixation-induced cell shape changes.

Animals↗
PubMed1987

Regulatory peptide immunocytochemistry at light- and electron microscopical levels.

Immunocytochemical techniques applied at both light- and electron microscopical levels are valuable in the study of regulatory peptide distribution in normal and diseased tissue, whether in the form of sections or whole cell preparations. Successful immunolocalization depends on adequate preservation of the peptide antigen and the tissue structure in which it resides; a suitably specific and sensitive labelled antibody detecting system. In general, peptides are stable molecules, most of which retain their antigenicity after conventional cross-linking fixation and tissue processing, allowing standard immunocytochemical methods to be used for light- and electron microscopy. Regulatory peptides are derived from precursor molecules and several 'families' of structurally similar peptides are now generally recognised. Region-specific antibodies may be needed to overcome problems of cross-reactivity or to identify a bioactive form in the presence of its precursor. Multiple co-localisation of different related and unrelated peptides in the same cell or even storage granule is now recognised and can be identified by immunocytochemistry.

Animals↗
PubMed1997

Fixation-dependent organization of core histones following DNA fluorescent in situ hybridization.

We have evaluated the effects of different DNA denaturation protocols commonly used in DNA fluorescent in situ hybridization (FISH) experiments on chromatin structure using indirect immunofluorescence. The use of antibodies to acetylated histones H3 and H4 demonstrates that the different procedures differ considerably in their extent of histone displacement. Procedures involving paraformaldehyde fixation were found to be compatible with the structural preservation of acetylated chromatin organization by indirect immunofluorescence. These results provide a basis for interpreting DNA FISH experiments aimed at determining chromatin organization of individual loci.

Acetylation↗
PubMed1992

MR imaging for measurements of ventricles and cerebral cortex in postnatal rats (H-Tx strain) with progressive inherited hydrocephalus.

As part of a wider investigation on brain structure and function in young rats with inherited hydrocephalus, this study was undertaken to develop a technique to monitor, and quantify, progressive changes in ventricle and cortical dimensions in hydrocephalic rats in vivo. Magnetic resonance images were obtained with a T1-weighted protocol and a multislice variant of the inversion recovery sequence which displayed optimal contrast between cerebrospinal fluid and brain parenchyma. Rats were imaged at 7, 14, and 21 days after birth by taking 10 contiguous 2.0-mm coronal slices. Area and distance measurements were taken from the displayed images, enabling calculation of ventricle volumes, cortical length, and cortical thickness. The lateral ventricle volume, which was already 100-fold larger than control at 7 days, increased to over 1000 mm3 at 21 days. The anterior-posterior length of the cortex was up to 27% larger in hydrocephalics and the cortical mantle was up to 75% thinner, particularly in the posterior cortex. Subsequent fixation and histological sectioning of two 21-day-old rats showed that the smaller dimensions, which were obtained from analysis of the sections, could be accounted for by shrinkage of the tissue during dehydration and wax embedding.

Aging↗
PubMed1998

Diameters of the main excretory ducts of the adult human submandibular and parotid gland: a histologic study.

In assessing new minimally invasive diagnostic techniques (duct endoscopy) and therapy (lithotripsy), it is of importance to know the true dimensions of the excretory ducts. Twenty-five ducts of the parotid gland and 20 ducts of the submandibular gland were examined histologically at different points of their anatomic course, and their in vivo diameters were evaluated with the use of a previously determined formalin-induced shrinking-factor. The mean diameter of Stensen's duct at four different points along its length ranged between 1.4 mm and 0.5 mm, with a maximum of 2.3 mm and a minimum of 0.1 mm, depending on the site. A narrowing at the middle of the duct was striking. In all preparations examined, the minimum width of the excretory duct was located at the ostium. In Wharton's duct the narrowest duct diameter was also identified at the ostium. The mean values for the duct diameters ranged between 1.5 mm and 0.5 mm. The largest duct diameter reached 2.2 mm; the smallest one, 0.2 mm. For diagnostic and therapeutic purposes, endoscopes, balloon catheters, and stone-extraction-baskets probably should, despite the extensibility of the duct, conform as much as possible to the physiologic duct widths. A diameter of 1.2 mm should be aimed at as an upper limit for these instruments. Our findings also suggest that, in the case of salivary stone lithotripsy, the best results will be achieved when the maximum size of stone fragments does not exceed 1.2 mm.

Adult↗
PubMed1999

Detection of oxidant producing-sites in glutaraldehyde-fixed human neutrophils and eosinophils stimulated with phorbol myristate acetate.

The aim of the present study was to detect oxidant-producing sites, and to elucidate their dynamic reorganization in human polymorphonuclear leukocytes (PMNs) fixed with glutaraldehyde which preserves cell structure. In biochemical analyses, the detectable O2- generation in unfixed PMNs upon stimulation with phorbol 12-myristate 13-acetate (PMA) in the presence of cytochalasin B was characterized by a lag period of approximately 10 sec followed by O2- production. The maximal rate reached was 3.18+/-0.07 nmol/min/l x 10(6) cells (mean+/-S.D.; n = 4) after 30 sec of stimulation. PMNs exposed to PMA and cytochalasin B followed by fixation with glutaraldehyde generated O2- without a lag period at a rate of 0.35+/-0.05 n mol/min/l x 10(6) cells (mean+/-S.D.) by the addition of NADPH as substrate to the cell suspension. In the cytochemical assays, we employed both cells exposed to PMA and cytochalasin B, and then fixed with glutaraldehyde followed by incubation in the cytochemical reaction medium (pre-fixed cells) and cells incubated in the medium containing PMA and cytochalasin B followed by fixation with glutaraldehyde (post-fixed cells). Oxidant reaction in the pre-fixed cells was detected by the addition of NADPH and FAD to the reaction medium. No oxidant-reaction product was seen in pre-fixed cells stimulated for 10 sec whereas the oxidant reaction was visualized in intracellular compartments of pre-fixed PMNs stimulated for 20 sec. The fact that the pre-fixed PMNs stimulated for 30 sec showed increased numbers of oxidant-producing structures compared to those seen in the pre-fixed cells stimulated for 20 sec, demonstrates that the amount of the reaction product and the number of oxidant-producing intracellular compartments increases between 20 and 30 sec after start of stimulation with PMA. These cytochemical results using the pre-fixed cells coincided with the findings obtained from the biochemical assays in the pre-fixed cells exposed to PMA and cytochalasin B. The oxidant reaction was observed in elongated tubular structures that were arranged in a radial fashion, and were associated with the plasma membrane in the pre-fixed PMNs, whereas post-fixed PMNs exhibited slender spherical or rod-shaped structures of various lengths. The present results indicate that the pre-fixed PMNs can be employed for elucidating the dynamic reorganization of oxidant-producing sites in human PMNs.

Benzoquinones↗
PubMed2004

Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment.

In this study, gel electrophoresis and capture enzyme-linked immunosorbent assay were used to assess the effect of formaldehyde treatment on the structural and immunological properties of bovine pancreatic ribonuclease A (RNase A). Prolonged incubation of RNase A in a 10% formalin solution leads to the formation of extensive intra- and intermolecular cross-links. However, these formaldehyde cross-links do not completely eliminate the recognition of RNase A by a polyclonal antibody. Comparative immunotitration of monomers, dimers, and oligomers greater than pentamers isolated from formalin-treated RNase A demonstrated that reduction of immunoreactivity due to intramolecular modifications prevails over the excluded volume effect of intermolecular cross-links. The latter only becomes important for intermolecular cross-links involving four or more molecules. The restoration of RNase A immunoreactivity during heating correlates with the reversal of formaldehyde cross-links if the incubation temperature does not exceed the denaturation temperature of the formalin-treated RNase A preparation. We conclude that formaldehyde cross-links stabilize antigens against the denaturing effects of high temperature, but the reversal of these cross-links is necessary for the restoration of immunoreactivity.

Animals↗