Use In Immunocytochemistry Research Articles

Commentary on a Classic JHC Article on the Development of Highly Sensitive Fluorochrome-labeled Tyramides for Immunocytochemistry.

This commentary reflects on the significance and impact of the highly cited companion article that was published in the Journal of Histochemistry and Cytochemistry in 1997 (Gijlswijk RPM et al. Fluorochrome-labeled Tyramides: Use in Immunocytochemistry and Fluorescence In Situ Hybridization. Journal of Histochemistry & Cytochemistry. 1997;45(3):375-382).

The selection and characterization of antibodies to minichromosome maintenance proteins that highlight cervical dysplasia

Background Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay. Methods Antibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples. Results Four antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 4 × 10 − 9 M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells. Conclusion In this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples.

Localization of the phase-related 6-kDa peptide (PRP) in different tissues of the desert locust Schistocerca gregaria—Immunocytochemical and mass spectrometric approach

A 6-kDa phase-related peptide (PRP) was recently identified from the hemolymph of the desert locust Schistocerca gregaria. Its presence in much higher concentrations in the crowd-reared (gregarious) phase than in the isolated-reared (solitarious) one suggests a role in phase polyphenism. However, when tested in a variety of classical bioassays, no activity could be found. We hoped that uncovering its site(s) of synthesis might yield hints as to possible functions. An antiserum was raised against the C-terminal 16 aa part of PRP for use in immunocytochemistry. No immunoreactivity was recorded in the fat body, midgut, or Malpighian tubules. The strongest positive immunostaining was observed in the follicle cells of the ovary and in the seminal vesicle tubes of the male accessory gland complex. Also, positive were a pair of large neurosecretory cells in the subesophageal ganglion, the storage part of the corpora cardiaca and some nerve fibers in the brain- and abdominal regions. An additional mass spectrometric analysis was successfully done in combination with a BLAST search to detect possible false positive staining. This confirmed the presence of genuine PRP in most of the immunopositive tissues. Additional experiments are needed to unravel the role of PRP.

Neutrophils Mediate Parenchymal Tissue Necrosis and Accelerate the Rejection of Complete Major Histocompatibility Complex-Disparate Cardiac Allografts in the Absence of Interferon-γ

A major feature of acute rejection of cardiac allografts is an intense mononuclear cell infiltration accompanied by interferon (IFN)-gamma production. In the current study we tested the role of IFN-gamma in acute rejection of allografts by comparing the histopathology of rejection in wild-type versus IFN-gamma-/- recipients of major histocompatibility complex-mismatched cardiac grafts. Wild-type recipients rejected the allografts at days 8 to 9 after transplant but rejection was accelerated 2 to 3 days in IFN-gamma-deficient recipients. During rejection in wild-type recipients, the allografts were heavily infiltrated with CD8+ T cells and other mononuclear cells. In contrast, allografts in IFN-gamma-deficient recipients had few T cells but an intense neutrophil infiltration accompanied by extensive graft parenchymal necrosis. No difference in expression levels of neutrophil chemoattractants including Groalpha/KC, MIP-2, GCP-2, and MIP-1alpha, was observed in allografts retrieved from wild-type and IFN-gamma-/- recipients. Depletion of neutrophils from IFN-gamma-deficient recipients delayed rejection until days 8 to 10 after transplant and restored the histopathology of acute allograft rejection to that observed in allografts rejected by wild-type recipients. These results indicate the potent regulatory properties of IFN-gamma during acute rejection directed at neutrophil infiltration into allografts and mediating graft tissue necrosis.

Specific detection of type III iodothyronine deiodinase protein in chicken cerebellar purkinje cells.

Because iodothyronine deiodinases play a crucial role in the regulation of the available intracellular T(3) concentration, it is important to determine their cellular localization. In brain, the presence of type III iodothyronine deiodinase (D3) seems to be important to maintain homeostasis of T(3) levels. Until now, no cellular localization pattern of the D3 protein was reported in chicken brain. In this study polyclonal antisera were produced against specific peptides corresponding to the D3 amino acid sequence. Their use in immunocytochemistry led to the localization of D3 in the Purkinje cells of the chicken cerebellum. Both preimmune serum as well as the primary antiserum exhausted with the peptide itself were used as negative controls. Extracts of chick cerebellum and liver were made in the presence of Triton X-100 to solubilize the membrane-bound deiodinases. Using these extracts in Western blot analysis, a band of the expected molecular weight ( approximately 30 kDa) could be detected in both tissues. Using a full-length (32)P-labeled type III deiodinase cRNA probe, we identified a single mRNA species in the cerebellum that was of the exact same size as the hepatic control mRNA (+/-2.4 kb). RT-PCR, followed by subcloning and sequence analysis, confirmed the expression of D3 mRNA in the chicken cerebellum. In this study we provide the first evidence of the presence of the D3 protein in a neuronal cell type, namely Purkinje cells, by means of immunocytochemical staining. We were able to detect a protein fragment corresponding to the expected molecular mass (30 kDa) for type III deiodinase by means of Western blot analysis. RT-PCR as well as Northern blot analysis confirmed the presence of D3 mRNA in the cerebellum.

Overview of antibody use in immunocytochemistry.

Immunocytochemistry, by definition, is the identification of a tissue constituent in situ by means of a specific antigen-antibody interaction where the antibody has been tagged with a visible label (). Cell staining is a powerful method to demonstrate both the presence and subcellular location of a particular molecule of interest (). Initial attempts to label antibodies with ordinary dyes were unsatisfactory because the label was not sufficiently visible under the microscope. A. H. Coons first introduced immunofluorescence in 1941, using specific antibodies labeled with a fluorescent dye to localize substances in tissues (). This technique was considered difficult, and its potential was not widely realized for nearly 20 yr. Early attempts focused on labeling the specific antibody itself with a fluorophore (see Chapter 6). The labeled antibody was then applied to the tissue section to identify the antigenic sites (direct method) () (see Chapter 15). Later, the more sensitive and versatile indirect method was introduced () (see Chapters 16–18). In this method, the specific antibody, bound to the antigen, was detected with a secondary reagent, usually another antibody that had been tagged with either a fluorophore or an enzyme.

Production and characterization of monoclonal antibodies to human interleukin-12.

Interleukin-12 is a cytokine that plays a central role in mediating cell mediated immunity via enhancement of a TH1 cell response. IL-12, unusually for a cytokine, has a heterodimeric structure made up of 35 kDa and 40 kDa subunits. The aim of this study was to produce and characterize monoclonal antibodies to recombinant human IL-12. Twenty-two monoclonal antibodies to IL-12 were successfully produced and subunit specificity determined using recombinant human IL-12 and chimeric murine/human IL-12. All antibodies were shown to react with the p40 subunit by ELISA and immunoblotting with three of the MAbs being found to cross-react with murine IL-12. Using two individual bioassays for IL-12, seven of the MAbs were shown to neutralize biological activity of IL-12. Ten of the antibodies were found to be of use in immunocytochemistry, reacting with LPS-stimulated peripheral blood monocytes. The approaches and difficulties encountered in characterizing antibodies to a heterodimeric cytokine are discussed together with possible reasons for the failure to generate antibodies to the p35 subunit.

Estradiol Stimulates Growth Hormone Production in Female Goldfish

The effects of estradiol (E2) on growth hormone (GH) production was investigated in gonad-intact female goldfish. It was first necessary to generate a specific antibody for use in immunocytochemistry, Western, and dot-blot analyses of GH production. To accomplish this, grass carp GH (gcGH) cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) and expressed inEcherichia coliand a specific polyclonal antibody to recombinant gcGH was generated in the rabbit. In Western blot, the anti-gcGH antibody specifically immunoreacted with recombinant gcGH, purified natural common carp GH, and with a single 21.5-kDa GH form from pituitary extracts of grass carp, common carp, goldfish, and zebrafish but not salmon, trout, or tilapia. Intraperitoneal injection of the recombinant gcGH enhanced the growth rates of juvenile common carp demonstrating biological activity of this GH preparation. Electron microscopic studies showed that the anti-gcGH-I antibody specifically reacted with GH localized in the secretory granules of the goldfish somatotroph. Using anti-gcGH-I in a dot-blot assay, it was found thatin vivoimplantation of solid silastic pellets containing E2(100 μg/g body weight for 5 days) increased pituitary GH content by 150% in female goldfish. In a second, independent study employing a previously characterized anti-common carp GH antibody for radioimmunoassay, it was found that E2increased pituitary GH content by 170% and serum GH levels by approximately 350%. The E2-induced hypersecretion of GH and increase in pituitary GH levels was not associated with changes in steady-state pituitary GH mRNA levels, suggesting that this sex steroid may enhance GH synthesis at the posttranscriptional or translational level. Previous observations indicate that GH can stimulate ovarian E2production. The present results show that E2can in turn stimulate GH production, indicating the existence of a novel pituitary GH–ovarian feedback system in goldfish.

Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization.

The peroxidase-mediated deposition of hapten- and fluorochrome-labeled tyramides has recently been shown to increase the sensitivity of immunofluorescence and fluorescence in situ hybridization techniques. We have evaluated a number of red, green, and blue fluorescent tyramides for detection of antigens in tissue sections and cytospin preparations and for the detection of hapten- and horseradish peroxidase-labeled probes hybridized in situ to cells and chromosomes. With few exceptions, all fluorescent tyramide-based methods provided a considerable increase in sensitivity compared to conventional immunofluorescence and FISH methods.

Spatial and temporal immunocytochemical analysis of drosulfakinin (Dsk) gene products in the Drosophila melanogaster central nervous system.

The spatial and temporal distribution of three peptides, DSK I, DSK II, and DSK 0, encoded by the Drosophila melanogaster drosulfakinin (Dsk) gene, have been examined in the central nervous system. DSK I and DSK II have a -RFamide C-terminus and are structurally similar to sulfakinin peptides; in contrast, DSK 0 contains -SFamide and is not structurally similar to sulfakinins. Antisera specificities were determined by the design of the antigens and confirmed by dot blot analysis and preincubation with peptides prior to their use in immunocytochemistry. The distribution of immunoreactivity suggests that all three DSK peptides are processed from the polypeptide precursor and expressed in many of the same cells. Expression was observed at all developmental stages with an increase in the level of staining and the number of immunoreactive cells as development progresses. Cells in the brain lobe, optic lobe, subesophageal ganglion, thoracic ganglia, and the eighth abdominal neuromere contain DSK-immunoreactive materials. Immunoreactive fibers project from some cells and extend into the brain and ventral ganglion with regions of extensive arborization. DSK 0 immunoreactivity provides initial evidence for the presence of a -SFamide peptide in neural tissue. The observed expression of DSK-immunoreactive materials throughout development in numerous cells of the central nervous system suggests that DSK peptides may serve as hormones, modulators, or transmitters involved in several functions.

Clustering of Na+ channels and node of Ranvier formation in remyelinating axons

Polyclonal antibodies were raised against a well conserved region of the vertebrate Na+ channel and were affinity purified for use in immunocytochemistry. Focal demyelination of rat sciatic axons was initiated by an intraneural injection of lysolecithin and Na+ channel clustering was followed at several stages of myelin removal and repair. At 1 week post-injection axons contained long, fully demyelinated regions. Na+ channel clusters appeared only at heminodes forming the borders of these zones, and at widely spaced isolated sites that may represent former nodes of Ranvier. Over the next few days proliferating Schwann cells adhered to axons and began to extend processes. Clusters of Na+ channels appeared at the edges of these structures. As the Schwann cells elongated, the clusters seemed to move with them, since they remained at edges and the distance between aggregates increased. Clusters associated with different Schwann cells ultimately approached each other and appeared to fuse. Na+ channels then coalesced further at these sites, forming new nodes of Ranvier in regions that previously were internodal. If Schwann cell proliferation were blocked by mitomycin, no new clusters of Na+ channels appeared within internodes. Under these conditions, heminodal clusters remained visible at 1 week postinjection, but by 2 weeks they were no longer detectable, suggesting that proliferating Schwann cells are required for their maintenance. Clusters at normal nodes of Ranvier remained. It is concluded that Na+ channel aggregation and mobility in demyelinated nerve fibers is controlled by adhering Schwann cells, resulting in the formation of stable new nodes of Ranvier during remyelination.

Production and characterization of an antibody against the human bone GLA protein (BGP/osteocalcin) propeptide and its use in immunocytochemistry of bone cells

We have generated and characterized an antibody that recognizes the C-terminal sequence of the propeptide of human bone GLA protein (BGP/osteocalcin) (amino acid −26 to −1, with +1 being the amino terminus of the mature protein). The range of sensitivity of the antibody, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.5–250 ng/ml. The antibody effectively recognized pro-BGP in cell layer extracts of transformed cells (KT-005), but did not recognize mature, propetide-less BGP in the medium from the same cultures. Strong labelling was obtained using this antibody in immunoperoxidase staining or immunofluorescence of both transformed and normal human bone cells in vitro. Monensin significantly altered the intracellular pattern of labelling in immunofluorescence studies, indicating that the recognized antigen was associated with the cellular secretory pathway. We also obtained a specific and strong staining of cells in tissue sections of human fetal bone. Antibodies against the mature protein strongly stained the mineralization front, but did not stain cells to any appreciable level. Newly embedded osteocytes were the predominant cell type stained in such material, suggesting that they may represent the major source of BGP in the intact tissue. These observations indicate that BGP synthesis is a late event in osteoblastic development and that antibodies generated against the propeptide sequence are a potentially powerful tool in the analysis of bone tumors and evaluation of osteoblastic differentiation.

Overview of antibody use in immunocytochemistry.

Overview of antibody use in immunocytochemistry.

Insect myotropic peptides: differential distribution of locustatachykinin- and leucokinin-like immunoreactive neurons in the locust brain

Locustatachykinin I is one of four closely related myotropic neuropeptides isolated from brain and corpora-cardiaca complexes of the locust Locusta migratoria. Antiserum was raised against locustatachykinin I for use in immunocytochemistry. It was found that the antiserum recognizes also locustatachykinin II and hence probably also the other two locustatachykinins due to their similarities in primary structure. Locustatachykinin-like immunoreactive (LomTK-LI) neurons were mapped in the brain of the locust, L. migratoria. A total of approximately 800 LomTK-LI neurons were found with cell bodies distributed in the proto-, deuto- and tritocerebrum, in the optic lobes and in the frontal ganglion. Processes of these neurons innervate most of the synaptic neuropils of the brain and optic lobes, as well as the frontal ganglion and hypocerebral ganglion. The widespread distribution of LomTK-LI neurons in the locust brain indicates an important role of the locustatachykinins in signal transfer or regulation thereof. As a comparison neurons were mapped with an antiserum against the cockroach myotropic peptide leucokinin I. This antiserum, which probably recognizes the native peptide locustakinin, labels a population of about 140 neurons distinct from the LomTK-LI neurons (no colocalized immunoreactivity). These neurons have cell bodies that are distributed in the proto- and tritocerebrum and in the optic lobe. The processes of the leucokinin-like immunoreactive (LK-LI) neurons do not invade as large areas in neuropil as the LomTK-LI neurons do and some neuropils, e.g. the mushroom bodies, totally lack innervation by LK-LI fibers. In some regions, however, the processes of the LomTK-LI and LK-LI neurons are superimposed: most notably in the central body and optic lobes. A functional relation between the two types of neuropeptide in the locust brain can, however, not be inferred from the present findings.

Production of an affinity-purified antibody against an aldehyde-treated neurofilament protein for use in immunocytochemistry

Fixation enhances cellular morphology and reduces loss of molecules during tissue processing. Antibodies against fixation-resistant epitopes are very useful, because they allow an immunocytochemical detection in tissue of better preserved morphology. However, fixatives can alter antigenicity and adversely affect the result of immunohistochemical procedures. To address this problem, this study examined the feasibility of generating antibodies to a paraformaldehyde-fixed antigen for use in immunohistochemical procedures. The large subunit of neurofilament proteins was selected for this study. Crude neurofilament proteins were isolated and separated by SDS-polyacrylamide gel electrophoresis. The large subunit of neurofilaments (NF-H) was electroeluted from the electrophoresis gel and exposed to paraformaldehyde, and used for immunization of a rabbit. The rabbit antiserum was affinity purified on CNBr-sepharose immobilized neurofilament proteins. On Western blots, the antibody reacted with the NF-H protein in a phosphorylation-dependent manner. In aldehydefixed cerebellum, the antibody strongly stained axons. In contrast, in alcohol-fixed cryostat sections the immunocytochemical detection was substantially reduced. The procedure presented in this study, involving a simple pretreatment of the immunogen, allows for the generation of an antibody that may be used in immunohistochemical studies where localization of the immunogen may be reduced or even lost by aldehyde fixation.

Characterization of a monoclonal antibody that specifically binds to choline phospholipids and its use in immunocytochemistry.

A monoclonal IgM (MC22-33F), raised in response to mouse embryonic dental papilla cells, was selected for further analysis on the basis of the unusual resistance of its epitope to detergent extractions and protease treatments of cell cultures. Binding of MC22-33F to cultured cells was abolished after either pre-treatment of the cells with phospholypase C or pre-incubation of the hybridoma culture supernatant with multilamellar phosphatidylcholine-containing vesicles. MC22-33F reacted with phosphatidylcholine, with the phosphatidylcholine analogue dimethylphosphatidylethanolamine, and with sphingomyelin immobilized on polystyrene surfaces or in thin-layer chromatograms. Crossreaction with other phospholipids was not observed. The surface of cultured epithelial cells was labeled by MC22-33F at sites of bleb formation. Combining immunostaining by MC22-33F and histochemical staining of cultured cells revealed codistribution of phospholipid-containing inclusions with either lysosomes or neutral fat droplets, and inhibition of lipid degradation by kanamycin resulted in a parallel accumulation of these inclusions and of neutral fats in the cytoplasm. Immunolabeling by MC22-33F of frozen mouse tissues was maximal in fat-storing and steroid-producing cells. Extracellular phospholipids present in calcifying cartilage septa strongly reacted with MC22-33F. This monoclonal antibody offers an interesting alternative to histochemical lipid stains for investigating fatty metamorphosis and extracellular lipid deposition under physiological and pathological conditions.

Monoclonal Antibodies to Substance P: Production, Characterization of Their Fine Specificities, and Use in Immunocytochemistry

Five hybrid clones secreting antibodies to the neuropeptide substance P have been obtained by somatic cell fusion of mouse myeloma cells with splenocytes from immunized mice of the Biozzi strain. To perform rapid and sensitive screening tests as well as to study the fine specificities of each monoclonal antibody, we developed a new enzyme immunoassay of substance P using acetylcholinesterase as label. All five monoclonal antibodies were directed to the C-terminal pentapeptide of substance P, especially to the Phe7 residue. They cross-reacted with neurokinin A and to some extent with neurokinin B but not with other nontachykinin mammalian peptides. One monoclonal antibody (SP 14) was used for immunocytochemical experiments in the rat spinal cord and spinal ganglion, both at the light and electron microscopic levels. A strong specific neurokinin-like immunoreactivity was observed in cell bodies, nerve fibers, and terminals, with a very low background staining. Finally, the affinities of several analogues of substance P for SP 14 monoclonal antibody were shown to be correlated with their biological activities, as measured by their hypotensive effects in vivo. These findings suggested a strong structural resemblance between the combining site of the antibody and that of the physiological substance P receptor.

Advantages of bispecific hybridomas in one-step immunocytochemistry and immunoassays.

A chemical selection procedure has been used to prepare a hybrid hybridoma cell line (P4C1) following fusion of two previously established hybridomas secreting antiperoxidase and antisubstance P, respectively. P4C1 secretes bispecific monoclonal antibody alongside the two parental antibodies, with no visible inactive heterologous heavy-light chain pairs. The bispecific monoclonal antibody is thus easy to purify in excellent yields. The advantage of its monovalency for one antigen and simultaneous binding of a marker enzyme has been explored for its potential use in competitive immunoassays. Its use in immunocytochemistry led to major improvements in sensitivity, signal-to-noise ratio, simplification of staining procedures, and ultrastructural preservation of subcellular elements. Particularly remarkable was that, unlike conventional procedures, the immunoreaction with the bispecific monoclonal antibody was homogeneously distributed across the entire thickness of a 50-micron section.

Specific antibodies against aspartate and their immunocytochemical application in the rat brain

An immunological approach to visualize aspartate in the rat brain was attempted by raising antibodies against this acidic amino acid. Using an adapted ELISA method, their specificity was tested by competition experiments between aspartate conjugated via glutaraldehyde to various protein-carriers and either non-conjugated aspartate or conjugated amino acids, preincubated with anti-aspartate antibodies. Their titer and specificity were found high enough to allow their use in immunocytochemistry which demonstrated the presence of a large number of aspartate-containing cell-bodies in many areas of the brain.

32] Characteristics of rabbit anti-PGH synthase antibodies and use in immunocytochemistry

Publisher Summary This chapter discusses the characteristics of rabbit anti-PGH synthase antibodies and its use in immunocytochemistry. Rabbit anti-PGH synthase sera when tested by standard Ouchterlony double-diffusion analyses show single lines of precipitation with samples of both solubilized sheep vesicular gland microsomes and pure PGH synthase; a zone of identity is present between the lines formed with microsomal and purified PGH synthases. When tested by immunoelectrophoresis, the purified cyclooxygenase shows an extended, but continuous, arc of precipitation that is because of the presence of multiple isoelectric forms of the enzyme. Immunocytochemistry provide convenient method for determining the location of prostaglandin synthesis in tissues and organs is to localize the PGH synthase. The rationale for this approach is that cells that synthesize prostaglandins generally do so beginning with endogenous arachidonate and, thus, must be able to convert arachidonic acid to PGH 2 via the PGH synthase.