Cryopreservation of cytological specimens for immunocytochemistry.

A method has been established for storage and preservation of cytological specimens in liquid nitrogen and further processing for immunocytochemistry as smears prepared from thawed cells or cryo-sections of frozen cell pellets. For the experiments cultured cells of a T-lymphoblastic leukemia cell line (ATCC CCL 119) and blood cells of the buffy coat of healthy humans were treated with a cryo-solution (fetal calf serum +5% dimethylsulfoxid) and after freezing stored in liquid nitrogen. Alternatively, cells preincubated with cryo-solution followed by suspension in fetal calf serum without cryo-additive were frozen and stored in liquid nitrogen for the production of cryo-sections. Indirect immunofluorescence and alkaline phosphatase--antialkaline phosphatase based immunoreactions were performed for the decoration of various surface antigens with a panel of monoclonal antibodies. All immunoreactions were repeated at least three times and the stored cell preparations were investigated after different periods of storage (up to four months). The immunoreactions of fresh cells in suspension (which were used as controls) were comparable with those of cryopreserved cells, e.g. cells on smears after thawing and on cryo-sections of cell pellets. The strongest immunoreactions were achieved on fixed cryo-sections. The maintenance of cell morphology of smears from cryopreserved cells was slightly better than of cells from cryo-sections. In our hands the preparation of cell pellets, which are suitable for the storage in liquid nitrogen and the production of cryosections, is a very useful method for immunocytochemical investigations of cytological specimens especially in situations where immunoreactions cannot be performed on fresh material.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity controls for immunocytochemistry

Antibodies have been in widespread use for more than three decades as invaluable tools for the specific detection of proteins or other molecules in biological samples. In spite of such a long experience, the field of immunocytochemistry is still troubled by spurious results due to insufficient specificity of antibodies or procedures used. The importance of keeping a high standard is increasing because massive sequencing of entire genomes leads to the identification of numerous new proteins. All the identified proteins and their variants will have to be localized precisely and quantitatively at high resolution throughout the development of all species. Consequently, antibody generation and immunocytochemical investigations will be done on a large scale. It will be economically important to secure an optimal balance between the risk of publishing erroneous data (which are expensive to correct) and the costs of specificity testing. Because proofs of specificity are never absolute, but rather represent failures to detect crossreactivity, there is no limit to the number of control experiments that can be performed. The aims of the present paper are to increase the awareness of the difficulties in proving the specificity of immunocytochemical labeling and to initiate a discussion on optimized standards. The main points are: (1) antibodies should be described properly, (2) the labeling obtained with an antibody to a single epitope needs additional verification and (3) the investigators should be required to outline in detail how they arrive at the conclusion that the immunocytochemical labeling is specific.     

An automated device for immunocytochemistry

A microprocessor controlled device for automation of immunoenzyme histological and cytological staining procedures is described. As the sequence of processing is controlled by a computer program, the apparatus is flexible and the possibility of technical errors is reduced.     

Modern methods for diagnostic immunocytochemistry

Immunocytochemistry has become an important diagnostic tool. The reliability or otherwise of the technique depends upon attention to detail and the optimization of every step in what is sometimes a complex technical procedure. This article outlines basic theory and practice for the most commonly used procedures. It highlights the problems of endogenous biotin staining and avidin–biotin-based detection methods. Labelled polymer based methods are explained and compared with other commonly used detection methods. Recent advances in high-sensitivity methodology are discussed.     

Tissue preparation for simultaneous flow cytometric quantitation of tumour associated antigens and DNA in solid tumours.

A multiparameter flow cytometric assay for the simultaneous study of tumour associated antigens (TAA) and DNA in fresh solid tumours was devised. Cell suspensions were prepared by disaggregating unfixed solid tumour samples mechanically over a stainless steel mesh. Indirect immunofluorescence was used to identify the TAA, and DNA was stained with propidium iodide. Cell morphology was well preserved, cell clumping was negligible, and high quality indirect immunofluorescence quality indirect immunofluorescence and DNA staining were obtained. The technique is simple, rapid, and reproducible. Multiparameter assays can be developed to study prognostic indicators such as membrane oncoproteins, receptors, and multidrug resistance in solid tumours. With a suitable panel of antibodies the technique might become an aid in the differential diagnosis and biochemical diagnosis of some solid tumours.     

Ultracryotomy of nerve-electroplaque synapses for immunocytochemistry

Summary In order to carry out ultrastructural immunocytochemical localization of acetylcholinesterase at cholinergic synapses, ultrathin frozen sections of aldehyde-fixed electric organs ofElectrophorus andTorpedo were obtained by a modified ultracryotome technique. Dry frozen sections, picked up with rabbit serum and negatively stained, revealed, in the postsynaptic regions of the neuro-electroplaque junction, the presence of numerous rod-like or pin-headed protrusions (30 × 40 Å) attached perpendicularly at 60 Å intervals to the hydrophobic lamina of the plasma membrane, forming a comb-like structure oriented toward the synaptic cleft. A possible correlation is suggested between this comb-like structure and the acetylcholine receptors observed by other authors with high resolution electron microscopy either after classical preparative techniques or after freeze-etching, of the postsynaptic membrane of vertebrate cholinergic synapses.     

Specificity and sensitivity of immunocytochemistry for detecting P-glycoprotein in haematological malignancies.

AIMS--To determine the optimal working conditions of the alkaline phosphatase-antialkaline phosphatase (APAAP) method to establish a specific and sensitive assay for the detection of low numbers of MDR positive cells in patients with hematological malignancies. METHODS--Three monoclonal antibodies (C-219, JSB-1, MRK-16) were used for the detection of P-glycoprotein (P-gp) in cell lines and in samples from 43 patients with haematological malignancies. The results of the APAAP method were compared with western blotting for specificity and sensitivity. RESULTS--Excellent correlation was obtained between optimised APAAP and western blotting, except in the case of multiple myeloma. JSB-1 seemed to be the more useful monoclonal antibody for the APAAP which was more sensitive than western blotting in its ability to detect single P-gp positive cells. CONCLUSIONS--Methods for P-gp detection, as defined by multidrug resistant (MDR) cell lines, are not necessarily optimal and specific for clinical samples and may lead to higher false positive and negative results, according to the conditions and the monoclonal antibodies used.     

The role of immunocytochemistry in diagnostic pathology.

This review suggests that immunocytochemistry in diagnostic pathology can be performed using relatively small panels of antibodies and that it should be reserved for situations in which, for one reason or another, the pathologist cannot exert his or her conventional diagnostic skills. Examples include the diagnosis of tumours the true nature of which is uncertain because of anaplasia or poor morphological preservation; the demonstration of small numbers of cells which are otherwise too rare to be recognised in conventionally stained preparations; and the immunophenotyping of non-Hodgkin's lymphomas. Recently progress has been made in the context of non-Hodgkin's lymphomas by the development of monoclonal antibodies that detect T and B cell associated markers in paraffin wax sections. Most of these reagents, however, recognise either lineage associated (but not lineage specific) variants of the leucocyte common antigen CD45, or antigens that are poorly characterised. A recent promising development has therefore been the demonstration that polyclonal antisera raised against the CD3/T3 T cell specific marker (purified by affinity chromatography) are suitable for staining T cells in paraffin sections. This approach will hopefully enable antibodies to be produced which react with other well defined white cell associated markers in routine biopsy material.     

Responses of odontoblasts to cavity preparation in rat molars as demonstrated by immunocytochemistry for heat shock protein (Hsp) 25

Responses of odontoblasts to cavity preparation in rat molars were investigated by immunocytochemistry for heat shock protein (Hsp) 25. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin. Confocal microscopy of Hsp 25-immunostained and rhodamine-labeled sections revealed that the immunoreactive odontoblasts were intensely labeled for phalloidin at the periphery of their cytoplasm and throughout their processes, but the reaction for phalloidin was limited within the inner half of the dentin. Cavity preparation caused an edematous reaction between the injured odontoblasts and predentin as well as a beaded swelling and successive destruction of the odontoblast processes. Immediately after cavity preparation, the odontoblasts beneath the edematous lesion showed an immunoreactivity for Hsp 25, which subsequently disappeared completely from the pulp-dentin border by 12 h after the operation. However, round cells without apparent cytoplasmic processes continued to be immunoreactive, suggesting the survival of a part of the odontoblasts against preparation stimuli. Numerous phalloidin-reactive but Hsp 25-immunonegative cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, probably categorized in a lineage of immunocompetent cells. By postoperative 72 h, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts, and suggest that this protein is a useful marker substance for differentiated odontoblasts.     

Lactotransferrin immunocytochemistry in Alzheimer and normal human brain.

Lactotransferrin (LF) expression was investigated immunocytochemically in postmortem brain tissues of normal controls and patients with Alzheimer's disease (AD). The antibody to LF stained some neurons weakly in young adult brains, but it stained many neurons as well as the glia of all types in elderly brains. LF expression was greatly up-regulated in both neurons and glia in affected AD tissue. It was very strongly associated with such extracellular pathological entities as diffuse and consolidated amyloid deposits and extracellular neurofibrillary tangles. In addition, it was identified in a minority of intracellular neurofibrillary tangles, neuropil threads, and degenerative neurites. LF is an iron scavenger and a complement inhibitor. Up-regulation may be a defense mechanism in AD-affected brain tissue.     

Transmissible ileal hyperplasia of hamsters. I. Histogenesis and immunocytochemistry.

Transmissible ileal hyperplasia (TIH) was experimentally induced in weanling hamsters, and the development of lesions was characterized. Ileal lesions developed in two phases: a hyperplastic phase which was detected by Day 10 and an inflammatory phase which began by Day 20. Hyperplasia began as focal lengthening of villi with expansion of crypt-type epithelium onto villus walls. Diffuse hyperplasia of distal ileum developed; dilated, tortuous crypts penetrated subjacent supporting tissues; but metastases were not seen. Inflammation began in association with focal or segmental necrosis of crypt epithelium, and crypt abscesses developed. Severe pyogranulomatous inflammation of the ileal wall, focal peritonitis, mesenteric lymphadenitis, and portal hepatitis were common in advanced lesions. Development of ileal lesions was closely correlated with accumulation of particulate antigen, detectable by immunofluorescence, in the cytoplasm of mucosal epithelial cells. Antigen was also detected in ileal granulomas, mesenteric lymph nodes, and liver. There was simultaneous development of serum antibody specific for intracytoplasmic antigen. These studies comfirm that mucosal hyperplasia is the primary lesion in TIH.     

Incubation chambers for the staining of ultrathin sections. An in situ technique especially suited for immunocytochemistry.

The preparation and use of incubation chambers for staining of ultrathin sections is described. As an example the immunogold staining is given which can be carried out in these devices in a more simple manner than normally possible, and with good results as shown by electron microscopy.     

Distribution of dengue-2 antigens by electron immunocytochemistry.

The distribution of dengue-2 antigens was studied in infected monkey kidney cells (LLC MK2) using an indirect, horseradish peroxidase-conjugated immunoglobulin technique. This procedure allowed both light and electron microscopic examination of serial-step sections of individual cells cut in a plane perpendicular to the monolayer. Both virion and nonvirion antigens were identified on the plasma membrane with this technique. A diffuse cytoplasmic reaction product was also present. The intensity and distribution of the cytoplasmic reaction product was related to disruption of the plasma membrane.     

Ethanol vapour-fixation of rat lung for immunocytochemistry investigations

Ethanol-vapour fixation of rat lung has been successfully employed in the immunocytochemical detection of the gastrin mucin antigen termed mucin 5AC without the need of additional antigen retrieval steps. This procedure gives good morphological preservation and provides all the benefits associated with the microscopic examination of inflated lung tissue. This simple fixation technique provides another option for use in immunocytochemical investigations of rodent lung and could be adapted for other species.     

Growth factor-induced proliferation of osteoblasts measured by bromodeoxyuridine immunocytochemistry.

Immuno-localization of BUdr was used to identify DNA synthesis in vitro in chicken embryonic bone cells stained positively or negatively for alkaline phosphatase activity. The results were similar to, but more sensitive than, our standard bioassay which assesses 3H-thymidine incorporation into DNA by liquid scintillation counting, and more rapid than autoradiographic localization of 3H-thymidine. SGF/IGF-II and bFGF stimulated cellular proliferation equally in ALP(+) and ALP(-) cells. In contrast, IGF-I and TGF-beta stimulate proliferation more in the ALP(-) than ALP(+) cells. The greatest increase in DNA replication of ALP(-) cells occurred following incubation with SGF/IGF-II or TGF-beta, and in the ALP(+) cells with SGF/IGF-II or bFGF. TGF-beta stimulated cellular proliferation at the lowest dose (1 ng/ml). The differential effect of the growth factors on each population of cells indicates that all these bone-matrix derived growth factors may play different roles in the local regulation of skeletal metabolism.     

Synaptophysin expression in neuroendocrine neoplasms as determined by immunocytochemistry.

Synaptophysin is an integral membrane glycoprotein originally isolated from presynaptic vesicles of bovine neurons. The authors have studied a wide spectrum of neuroendocrine (NE) neoplasms by immunofluorescence microscopy on cryostat sections of freshly frozen tissues using a monoclonal antibody to this protein (SY 38). Without exception, they found the identical--or a very similar--protein expressed in all neuroblastomas, ganglioneuroblastomas, ganglioneuromas, pheochromocytomas, and paragangliomas studied. In these "neural" type NE neoplasms, synaptophysin was coexpressed with neurofilament proteins. Synaptophysin was also demonstrated in NE neoplasms of "epithelial" type in which it was predominantly coexpressed with cytokeratins and desmoplakin. It was invariably found in all variants of islet cell neoplasms and in all medullary thyroid carcinomas. Synaptophysin was also demonstrated in several adenomas of the hypophysis and parathyroids, in the majority of carcinoids of the bronchopulmonary and gastrointestinal tracts, and in many, though not all, NE carcinomas of the same sites, and of the skin. Conversely, SY 38 did not immunostain any of a large number of benign and malignant non-NE epithelial neoplasms; nor was any immunostaining obtained in a group of mesenchymal tumors. It is remarkable that SY 38 did not immunostain a number of malignant melanomas, including several that were immunostained for neuron-specific enolase (NSE) and several neuropeptides. Parallel studies conducted on conventionally fixed, paraffin-embedded tissue sections immunostained by the use of the avidin-biotin complex technique yielded very similar results. The findings indicate that synaptophysin is expressed in the whole range of NE neoplasms without detectable relation to the expression of other NE markers such as NSE, serotonin, and neuropeptides. Nor could the expression of synaptophysin by these tumors be correlated with their epithelial and/or neural cytoskeletal characteristics, their clinical aggressiveness, or the presence or absence of endocrinologic abnormalities. While the consistent expression of synaptophysin by the "neural" type of NE neoplasms would seem predictable its presence in diverse benign and malignant NE tumors of "epithelial" type is remarkable. It is concluded that synaptophysin is a significant as well as novel NE marker, and the use of antibody SY 38 as a broad range marker for the study and diagnosis of NE neoplasms is proposed.