Simultaneous immunohistochemical demonstration of antigen expression and 5-bromodeoxyuridine incorporation in plastic embedded sections

Summary In this study a double immunohistochemical staining procedure is described for the simultaneous demonstration of antigen expressing cells and replicating cells in rat thymus. As markers for cell surface antigen expression a monoclonal antibody against Ia-expressing cells (His 19) and a monoclonal antibody against cells of the monocyte-macrophage lineage (ED2) were used. Replicating cells were demonstrated by the incorporation of 5-bromodeoxyuridine (BrdUrd). Tissue pieces were fixed in a periodate-lysine-paraformaldehyde fixative and embedded in glycol methacrylate. To demonstrate Ia-expressing cells or ED2-positive macrophages in plastic embedded sections a digestion with trypsin is necessary. The staining procedure was applied sequentially and was performed with a peroxidase and an alkaline phosphatase labeled reagent yielding respectively a brown and a blue reaction product. Results with this staining procedure on plastic embedded sections of rat thymus, an organ with a high DNA synthesizing capacity, showed incorporation of BrdUrd predominantly in the cortex. ED2-positive macrophages were only found in the cortex. The la-positive epithelial reticular cells demonstrated extremely well their stellate form.     

Postembedding immunohistochemical demonstration of antigen in experimental polyarthritis using plastic embedded whole joints

Summary A method is presented for the immunohistochemical demonstration of antigens in whole undecalcified joints of small laboratory animals. With this method of tissue preparation, involving embedding in a medium mainly based on 2-hydroxyethyl methacrylate, preservation of antigenicity is satisfactory. Antigens can be demonstrated in 2 m sections by either immunofluorescence or immunoperoxidase and an indirect technique. Therefore in addition to the morphological analysis of joint alterations in experimental polyarthritis, there is now an opportunity to trace the inciting antigen and to study in parallel the enzymatic equipment of the cells involved, using consecutive sections from a single block of tissue.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 54, C2     

Non-random incorporation of 5-bromodeoxyuridine in rat cell DNA

Secondary cultures of rat embryo cells were exposed for 24 hrs. to 10-⁷M [³H] thymidine (TdR) or 10^?7M [³H]5-bromodeoxyuridine (BrdU) in order to localize and compare the distribution of the isotopes in DNA. DNA was extracted, sheared, and centrifuged to equilibrium through neutral and alkaline CsCl density gradients. The DNA band from each gradient type was separated into a “heavy” and “light” fraction, and DNA-DNA reassociation hybridizations were performed on each sample. Renaturation profiles revealed that each fractionated DNA sample was representative of the complete rat cell genome, except for the “light” [³H]BrdU-DNA prepared by centrifugation through alkaline CsCl gradients. This fraction was predominantly depleted of labeled late repetitive and intermediate sequences. Uncentrifuged rat DNA was sequentially fractionated during reassociation into rapidly, intermediate, and slowly reassociating sequences by hydroxyapatite chromatography. Relative specific activities of each component revealed a non-uniform distribution of [³H]BrdU moieties as compared to [³H]TdR. These results suggest a nonrandom incorporation of 10^?7M BrdU into rat cell DNA sequences.     

Immunohistochemical demonstration of various antigens in tissues embedded in plastic

In the course of a chronic experimental study of polyarthritis in rats, we have established a plastic embedding technique which allows the processing not only of soft tissues but also of tissues such as cartilage, intraarticular ligaments, and even undecalcified whole rat joints and provides broad survival of enzymes and antigens. Preservation of morphological details and the precision of antigen localization are superior to those seen in cryostat and paraffin sections. The method seems to have much diagnostic and investigative value, especially in arthritis research.     

Enzymatic determination of nonrandom incorporation of 5-bromodeoxyuridine in rat DNA.

Secondary cultures of normal rat embryo cells were synchronized by a double thymidine block and pulsed with 10(-7) M 5-[3H]bromodeoxyuridine (BrdUrd) OR 10(-7) M[3H]thymidine during an entire S phase (7.5 h). To examine the pattern of [3H]thymidine, DNA was immediately extracted and purified at the completion of the S phase, CsCl density gradient centrifugation revealed that substitution for thymine by bromouracil was less than 7%. Single-strand specific nucleases obtained from Aspergillus oryzae and Neurospora crassa were allowed to react with native and partially depurinated (24-29%) [3H]BrdUrd-labeled rat DNA samples, and the products were assayed by hydroxylapatite column chromatography. Approximately 4-6% of the native, nondepurinated rat DNA was hydrolyzed by both nucleases. However, 24-28% of the partially depurinated, [3H] thymidine-labeled rat DNA was hydrolyzed by both enzymes as determined by loss of mass as well as radioactivity. Whereas comparable levels of depurinated, [3H]BrdUrd-labeled DNA were physically hydrolyzed by both nucleases, nearly 65% of the radioactivity was not recovered. Native, as well as depurinated, enzyme-treated DNA samples were sequentially and preparatively reassociated into highly repetitive, middle repetitive, and nonrepetitive nucleotide sequence components. The absolute and relative specific activities of each subfraction of native [3H]thymidine-labeled DNA were comparable. [3H]BrdUrd was differentially concentrated in the middle repetitive sequences as compared to other reiteration frequency types. When depurinated, nuclease-treated DNA samples were similarly fractionated, [3H]thymine moieties were uniformly distributed thoughout all sequences. However, a differential loss of [3H]BrdUrd moieties was detected predominantly from the middle repetitive nucleotide fraction. Melting profiles of the renatured DNA samples were characteristic of each respective DNA subfraction regardless of isotopic precursor. These results suggest that [3H]BrdUrd may be differentially incorporated into A + T rich clusters of rat DNA, especially in the moderately repeated chromosomal elements.     

Silver-staining specificity in metaphases after incorporation of 5-bromodeoxyuridine (BUDR)

Summary Silver staining is reported to be reduced in chromatin substituted by BUDR. This quenching effect allows for the demonstration of replication patterns and differential chromatid staining. Though the differentiation, as compared to other staining techniques, is of inferior quality, it is of theoretical importance concerning the effect of BUDR incorporation into chromatin.     

Effect of 5-bromodeoxyuridine on incorporation of RNA precursors in sea urchin embryos

Exposure of early sea urchin embryos to 5-bromodeoxyuridine (at concentrations up to 100 μg per ml) severely decreases the uptake of exogenous ³H-uridine into RNA. However, the actual gross rate of DNA or RNA synthesis in these embryos appears not to be affected by the presence of 5-bromodeoxyuridine.     

A comparison of 5-bromodeoxyuridine and thymidine incorporation into fertilized sea urchin eggs

³H-Thymidine (³H-TdR) and ³H-5-bromo-2-deoxyuridine (³H-BUdR) are first incorporated into newly fertilized Strongylocentrotus purpuratus eggs 30 min after fertilization, which is about the time of pronuclear fusion. An initial incorporation of ³H-BUdR immediately following fertilization has been previously reported. This was found to be due to a radioactive impurity present in samples of ³H-BUdR stored in aqueous solutions. ³H-TdR stored in water, or ³HBUdR stored in 50% ethanol, did not contain this impurity.     

Differences between diploid and transformed human cells in the incorporation of 5-bromodeoxyuridine into DNA

In diploid human cells, the DNA precursor pool equilibration times for exogenous thymidine are about twice those for the thymidine analogue 5-bromodeoxyuridine (BUdR); in cells that were either transformed chemically or derived from malignant tumours, the pool equilibration times are the same for thymidine and 5-bromodeoxyuridine and are closer in value to the shorter (bromodeoxyuridine) times of the diploid cells. Thymidine, if present in the culture medium with BUdR, is incorporated into DNA preferentially in diploid cells (by 2 or 3 to 1). Discrimination against bromodeoxyuridine is evident within 2 h of incubation of the two precursors with diploid cells, but is not observed even after 24 h in any of the transformed cell lines tested. Experiments were performed to test the effect of inhibitors of the mammalian DNA polymerases alpha (N-ethylmaleimide) and beta (incubation of cells at 45 °C) upon the ability of cells to synthesise DNA and to incorporate thymidine preferentially when present with equimolar BUdR. In diploid cells, overall in vivo DNA synthesis is more sensitive to N-ethylmaleimide and more resistant to 45 °C treatment than is DNA synthesis in the transformed cell lines. N-Ethylmaleimide decreases the capacity of diploid cells to discriminate against BUdR, whereas heating increases it. Transformed cells treated with N-ethylmaleimide remain unable to discriminate against BUdR; some transformed lines, when heated at 45 °C, become less incapable of such discrimination.     

Simultaneous immunohistochemical demonstration of vasoactive intestinal polypeptide and its receptor in human colon

Summary The present study reports an immunohistochemical approach for localizing the immunoreactivity of vasoactive intestinal polypeptide (VIP) receptor in the human colon, by using a monoclonal antibody which recognizes the VIP-receptor of a human colonic adenocarcinoma cell line. Simultaneous demonstration of immunoreactive VIP-receptor of a human colonic adenocarcinoma cell line. Simultaneous demonstration of immunoreactive VIP-receptor and VIP was achieved by a double-labelling procedure employing immunogold silver staining for VIP-receptor, and a biotinylated secondary antibody followed by streptavidin-Texas Red, to visualize VIP.The immunoreactive VIP receptor was found at two locations receiving dense VIP innervation: myenteric ganglia and mucosal epithelium.Epithelial cells displayed intense labelling at the basolateral membrane, which confirmed earlier binding studies on fractionated membranes. A small number of enteroendocrine cells was also recognized by the VIP-receptor antibody. Smooth muscle and cells of the immune system were not stained by the monoclonal antibody, indicating that it recognized an epitope not common to VIP-receptors of all locations.Thus, the immunohistochemical approach of VIP-receptor localization differs from autoradiography in (a) precise cellular localization, (b) possibility of simultaneous demonstration of receptor and ligand immunoreactivity, and (c) selectivity to a certain receptor population which, however, is presently not fully characterized.     

Effects on chromosomal proteins in sister chromatid differentiation by incorporation of 5-bromodeoxyuridine into DNA

When fixed metaphase chromosomes of human lymphocytes grown in the presence of BrdUr for two cell cycles were stained with amino group-specific 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) after a previous extraction of DNA, sister chromatids showed a light-independent differential staining. Although more faintly differential, a similar staining pattern being just the reverse of the DNA-specific DAPI pattern was obtained without prior removal of DNA. We conclude that the chromatid containing bifilarly BrdUr-substituted DNA has a higher protein content, at least after fixation, than the chromatid containing unifilarly BrdUr-substituted DNA. Possibly, a higher degree of BrdUr substitution leads to a tighter binding of chromosomal proteins. In line with this suggestion we found a markable difference in DNA extractability of BrdUr-containing and normal cytological preparations.     

Optimal parameters for the histochemical demonstration of acetylcholinesterase in plastic sections of rat brain

A modified method for improved preservation and optical resolution of acetylcholinesterase (AChE)-containing structures in adult rat brain is described. Optimal tissue preparation included fixation in paraformaldehyde 4%, glutaraldehyde 0.1%, and sucrose 7% in 0.1M Sorensen's phosphate buffer, pH 7.4, rinsing in buffer 50 mM with respect to NH4Cl and 2% with respect to sucrose, acetone dehydration, vacuum infiltration with LKB Historesin, and polymerization at 4 C, overnight incubation of 10 microns sections at 37 C in the AChE histochemical reaction mixture and silver intensification according to Hedreen et al. Demonstration of AChE enzyme activity in the cholinergic projection from the rat basal forebrain to the ipsilateral hippocampus exemplifies the usefulness of the technique. The method provides an excellent demonstration of AChE-positive axonal processes and enables the pharmacohistochemical visualization of cholinergic neurons. This procedure offers a convenient method for analysis of cholinergic neurons that avoids potential artifacts inherent in other AChE histochemical procedures.