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The modification consists of the replacement of alcohols of increasing concentration by glycerols. This results in a shortening of tissue treatment, improvement of the preparation quality due to the full exclusion of the alcoholic tissue shrinkage and cell structure deformation as well as alcohol saving.
A differential staining method of myelinated fibers and nerve cell bodies applicable to whole blocks of mammalian central nervous tissue is described. Experimental material fixed by perfusion or necropsy material fixed in block can be used. Blocks of 2 mm in thickness were obtained with a vibratome, immersed in 50% ethanol for 7 h and stained for 1 week in the following solution: 0.3% thionine, 5% formaldehyde and 5% acetic acid. After the staining period the blocks were washed in distilled water, dehydrated through graded alcohols, cleared in butyl acetate and infiltrated in paraffin. Sections of 10 microns in thickness were obtained, attached to slides, dewaxed in xylene and coverslipped with mounting media in the usual manner.
A method is described, combining immunohistochemistry and histological staining methods in the same section, for the identification of ACTH- and STH-producing cells in formalin-fixed, routine processed human pituitary tissue. The results indicate that this method is suitable to employ in practice.
Cytologic preparations provide a rapid, simple method for intraoperative diagnosis of central nervous system (CNS) lesions. Details of cellular morphology are defined sharply, avoiding artifacts often introduced by the frozen section technique. In 100 neurosurgical biopsies performed between 1984 and 1986, touch preparations and cryostat (frozen) sections were made at the time of surgery for preliminary intraoperative diagnosis. To assess the accuracy of each of the diagnostic methods used independently, slides obtained with each of the two techniques were later reviewed retrospectively with appropriate clinical and radiological data, but without knowledge of the final neuropathological diagnoses. When compared with the final diagnoses, intraoperative diagnoses were confirmed in 95 cases. The diagnoses based on cytologic and frozen section techniques were compared with the final diagnoses based on paraffin sections. Touch preparation diagnosis was confirmed by paraffin sections in 76 cases; in 18 additional cases a clinically useful, but nonspecific diagnosis (benign versus malignant, glial versus nonglial) was established by touch preparation. In five cases with firm or rubbery tumors, insufficient cells were imprinted for reliable evaluation, and no definitive diagnoses could be made. Specific cryostat diagnoses were confirmed by paraffin sections in 88 cases; nonspecific diagnoses were made in 11 cases. A single incorrect diagnosis was obtained with each technique. When the two techniques were used together, a specific and accurate diagnosis was achieved in 95 cases. Touch preparations were superior to frozen sections for evaluating soft or highly cellular tumors and for preliminary diagnosis from a minute surgical specimen (i.e., stereotactic biopsy).(ABSTRACT TRUNCATED AT 250 WORDS)
Nuclear suspensions were prepared from archival material of cancer biopsies. DNA content analysis by flow cytometry was performed after storage at -80 degrees C for 24 h and 6 months, compared to 24 h storage at +4 degrees C. No significant variations in CV or DNA indices were observed. Repeated freezing and thawing did not reduce the DNA histogram quality.
The oxidative cross-linking effect exerted by OsO4 on lipids and the blackening of osmiophilic structures resulting from reduction of OsO4 constitute long-known phenomena. Nevertheless, osmic acid has rarely been used for lipid staining as various even non-lipid-containing tissue structures are more or less intensively blackened by OsO4. If one treats osmicated tissue sections with certain oxidants (e.g. ammonium persulfate) in aqueous solution, then all the black OsO4 reduction products which are exposed to the aqueous phase are oxidized and thereby bleached. This applies e. g. to all proteins and polar (hydrophilic) lipids. Only apolar lipids retain their more or less black color and can be stained subsequently with Sudan dyes. Based on this principles, a new procedure has been developed which is effective on paraffin sections obtained after previous block osmication. This OBS procedure can also be used for very precise localization of appropriate lipid structures as can be demonstrated in cases of pathologic lipid deposits in an-alpha- and in a-beta-lipoproteinaemia, in type III hyperlipoproteinaemia, and in diverse other dyslipoproteinaemias as well.
The PAP procedure was compared with the peroxidase labelled antibody sandwich method and was found to be at least 20 times more sensitive. Background staining was reduced by the addition of normal swine serum to all the immune sera or by pretreating sections with it at the beginning of either method. The PAP procedure could be effectively reduced to a period of 1 hour or less.
Monoclonal antibodies (MoAbs) to monocytes/macrophages usually detect surface membrane antigens that are reactive only on cryostat sections of unfixed tissue. Such sections do not afford structural preservation conducive to satisfactory morphological characterization of various cell types. Moreover, in pathology laboratories the indication for immunohistochemical analyses often arises after routine preparation of the biopsy material has been carried out. Another difficulty is that the available MoAbs have shown a considerable degree of detection heterogeneity, including various dendritic cells (DC) and also various cell types beyond the monocyte-derived lineage. Conversely, the L1 protein is a formalin-resistant myelomonocytic antigen for which a MoAb is now available; and this cytoplasmic marker has a remarkably restricted distribution within the monocyte-derived cell lineage. By paired staining for L1 and S-100 protein, macrophages and DC of the interdigitating or Langerhans type can be visualized simultaneously in routine formalin-fixed paraffin sections. Such immunohistochemical staining is useful for the mapping of reactive tissue elements and characterization of histiocytic neoplasms. Interdigitating and Langerhans' DC can, in addition, be demonstrated in paraffin sections of ethanol-fixed tissue by means of MoAb to HLA-DR. MoAbs are also available for detection of follicular DC in such material. Visualization of the latter cells is of interest for the evaluation of lymph nodes from patients infected with the AIDS virus.
PURPOSE: The determination of Her2/neu over-expression has become necessary for the selection of breast cancer patients for Herceptin therapy. Immunohistochemistry in combination with FISH is currently the most used method for this detection. The objective of our study was to compare the use of multiparameter DNA flow cytometry (MP-FCM) for this assessment with immunohistochemistry (IHC). For this purpose the number of positive cells as well as the intensity of the immunofluorescence signal as determined by MP-FCM were compared with IHC scores. MATERIALS AND METHODS: Single cell suspensions from 110 breast cancers were analysed on a DAKO Galaxy flow cytometer after simultaneous (immuno)labeling for cytokeratin, Her2/neu and DNA (propidium iodide). Next to ploidy and proliferative activity (S-phase fraction), the fraction of Her2/neu-positive epithelial cells was determined. In 25 cases the intensity of the immunofluorescence signal was also determined by MP-FCM in the total cytokeratin+ fraction as well as the cytokeratin+/Her2/neu+fraction. Immunohistochemistry for Her2/neu was done on paraffin sections of all cases using the same polyclonal Her2/neu antibody in a SABC-peroxidase procedure after heat-induced antigen retrieval. RESULTS: Her2/neu overexpression as determined by IHC showed a score of 0 in 30%, 1 in 25%, 2 in 26% and 3 in 19% of the cases. There was a good correlation when comparing the fraction of Her2/neu-positive epithelial cells as determined by MP-FCM and semiquantitative IHC scores (r = 0.89, p < 0.0001). No correlation was found between the IHC-Scores and the intensity of the Her2/neu- staining in the total epithelial compartment as measured by MP-FCM. However, when restricting the intensity-determination to the Her2/neu-positive epithelial cells, a significant correlation was found between MP-FCM and IHC scores (r = 0.72, p < 0.0001). CONCLUSION: The results of the current study show that MP-FCM can be a good alternative for the objective quantification of Her2/neu.
A monoclonal antibody (MAb) (Z3A5) against spike protein subunit of bovine coronavirus (BCV) reacted with the virus in formalin-fixed intestines in an immunoperoxidase test. We found an 88% correlation between immunohistochemistry with Z3A5 and in situ hybridization with a BCV nucleoprotein cDNA probe. MAb Z3A5 reacted with 90 BCV isolates from the United States and was an effective reagent for the diagnosis of BCV.
We describe the immunocytochemical localization of four peroxisomal enzymes by light microscopy in human liver and kidney processed routinely by formalin fixation and paraffin embedding. Monospecific antisera against catalase and three enzymes of peroxisomal lipid beta-oxidation (acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase) were used in conjunction with either the indirect immunoperoxidase method or the protein A-gold technique followed by silver intensification. The sections of formalin-fixed paraffin-embedded tissue had to be deparaffinized and subjected to controlled proteolysis in order to obtain satisfactory immunostaining. Under the conditions employed, peroxisomes were distinctly visualized in liver parenchymal cells with no reaction in bile duct epithelial or sinusoidal lining cells. In the kidney, peroxisomes were confined to the proximal tubular epithelial cells with negative staining of glomeruli, distal tubules and collecting ducts. A positive immunocytochemical reaction was obtained even in paraffin blocks stored for several years. The method offers a simple approach for detection of peroxisomes and evaluation of their various enzyme proteins in material processed routinely in histopathology laboratories and should prove useful in the investigation of the role of peroxisomes in human pathology for both prospective and retrospective studies.
Staining of 1000 consecutive liver biopsies with orcein showed positive reaction in the cytoplasm of ground-glass hepatocytes in 18 of the biopsies. On new sections of the 18 orcein positive biopsies immunoperoxidase staining was performed in order to demonstrate hepatitis B-surface antigen (HBsAg). After destaining the same sections were stained with orcein. All biopsies showing positive orcein staining showed positive immunoperoxidase reaction. The number of positive cells was in biopsies with less than 20 positive cells per mm2 biopsy larger using the immumoperoxidase staining than with orcein staining. Further the staining contrast was more pronounced. Immunoperoxidase staining thus seems more sensitive than orcein staining for demonstrating HBsAg in liver tissues. Orcein stains HBsAg in liver tissue even though the antigen determinants are blocked by antibodies.
Eastern equine encephalomyelitis (F.EE) virus was detected in infected formalin-fixed horse and emu tissues and in infected chicken embryo fibroblasts. Results of in situ hybridization using a digoxigenin-labeled 40-base DNA probe complementary to a conserved region of the EEE virus RNA compared favorably with results of both virus isolation and serum neutralization tests. This technique may be useful for diagnosis of EEE virus infection in various animal species, especially when fresh tissues are not available for analysis, and also will provide a means for studying the involvement of alphaviruses in pathogenesis studies.
The specificities of carbonic anhydrase isoenzyme C (CA C) and glial fibrillary acidic (GFA) protein as immunocytochemical markers for different glial cell populations in human brain and retina were studied using indirect immunofluorescence and peroxidase-antiperoxidase complex methods. With antibodies against CA C, only those cerebral cells that were morphologically oligodendrocytes and Müller cells of the retina showed positive immunostaining reaction, whereas antibodies against GFA protein selectively labeled cerebral astrocytes and a part of the glial cells and fibers in the inner layers of the retina. In double labeling, when both glial cell markers were successively localized in the same cerebral tissue sections, GFA protein immunofluorescence was never found in the immunoperoxidase-stained CA C-positive cells, which further supports the oligodendrocyte-specificity of CA C in human brain.
52 specimens from 34 patients with multiple myeloma and 3 cases with reactive plasmacytosis were stained by the 2-step labelled or 3-step unlabelled immunoperoxidase (IP) technique for demonstration of intracellular immunoglobulin in order to test the utility of these methods as staining methods in routine diagnostic work. The specificity of the methods was high as agreement between IP staining and serumanalysis for M-component was achieved in 27 out of 28 cases, where complete serumanalysis were available. Interpersonal variation in interpretation of the staining results was low. With optimal dilutions of antisera the evaluations of the examiners were concordant in 51 out of 52 specimens. By comparing the different tissue specimens the best result was obtained with iliac bone biopsies both concerning IP staining and morphological preservation. No difference was observed between results obtained by the 2-step labelled and 3-step unlabelled IP techniques.
We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.
An avidin-biotin complex immunohistochemistry technique was developed to detect Haemophilus parasuis serovar 5 in experimentally infected 18-21-day-old conventional pigs, using a rabbit polyclonal antiserum. Seven of 10 intratracheally inoculated animals developed a low to medium degree of fibrinous polyserositis; meninges and pleura were the most severely affected areas. Haemophilus parasuis was recovered from 9 of 10 pigs; in 2 of them H. parasuis was isolated from tracheal swabs only. Positive immunohistochemistry results, mainly observed as free bacteria or bacteria within inflammatory cell cytoplasm in the fibrinopurulent exudate, were observed in 8 of 10 animals. Cross-reactivity with Actinobacillus pleuropneumoniae was detected but not with other gram-positive and gram-negative bacteria tested. This immunohistochemistry technique seemed to be at least as sensitive as microbiologic cultures and could be useful in studies of pathogenesis and retrospective diagnosis. However, cross-reactivity with A. pleuropneumoniae means that positive immunohistochemistry results in lung tissue from field cases would be dubious.
60 intracranial tumors have been studied immunohistochemically to determine the proliferation rate by staining for the monoclonal antibody KI-67, which recognizes a nuclear antigen expressed by cells in proliferation. In gliomas a clear correlation of stained nuclei to the histologically determined degree of malignancy was found: slow growing astrocytomas and oligodendrogliomas had an average proliferation rate of 1%, more malignant forms of 7-10%. Glioblastomas were found to have a growth fraction of 15%. Metastases had an even higher rate of 20% proliferating cells. In meningiomas the proliferation rate was mainly about 1%, but in three cases it was between 5% and 7%. Whether this is indicative for a higher risk of tumor recurrence, remains to be correlated to the clinical course. Hemangiopericytomas had a proliferation rate of 9% and 16%, respectively, the latter recurring within four months. It may be concluded from the results of this study, that investigation of intracranial tumors with KI 67 may be of prognostic value and can possibly contribute to an individualized tumor therapy.