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Distribution of a growth inhibitor in mammary tissue of cow and mouse was described by means of immunogold-silver stained semithin Lowicryl- and cryosections. In pregnant and lactating state of the tissue, remarkable amounts of the growth regulating protein of 1r = 13,000 could be detected mainly in epithelial cells.
In this technique for determining the electrophoretic phenotype of alpha 1-antitrypsin in dried blood or serum specimens, the adsorbed material is eluted with a concentrated solution of dithiothreitol, focused on polyacrylamide thin-layer gel, and made visible with silver stain. With this staining technique all normal and pathological alpha 1-antitrypsin phenotypes can be detected. The procedure is relatively simple, inexpensive, and suitable for use in large-scale screening for alpha 1-antitrypsin deficiency in selected populations.
The authors present a new technique for the analysis of the cerebro spinal fluid (C.S.F.) proteins: silver stain after isoelectric focusing with possibility of immunofixation. This technique is simple and results are obtained after six hours only after sample deposition. If immunofixation is used a further 24 hours washing is necessary. It is highly sensitive and bands containing 25 ng of proteins can be clearly distinguished so that small volumes (10 microl. at mean) of unconcentrated C.S.F. can be used. With this technique abnormal distinct bands of IgG specificity are seen in the majority of central and peripheral nervous system inflammatory disorders. No distribution of these bands, particularly in the more cathodal zone, seems to be specific of any disease including multiple sclerosis (M.S.). The abnormal IgG bands in M.S.C.S.F. seem to have their counterpart in the serum. These results need further verification. The usefulness of this technique for research purposes is stressed. The protein pattern obtained must be interpreted in the light of the concepts of genetic polymorphism, microheterogeneity and heterogeneous mode of antibody production.
The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient-gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional "macro"-method with self-cast SDS-polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 microL sample volumes and an 8-25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS-PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha-1-antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS-PAGE and staining allows easy standardization of urine SDS-PAGE among clinical routine laboratories.
In this study, BrdUrd labeling of S-phase cells in the small intestine and testes was accomplished using microwave irradiation. In this way crypt cells, spermatogonia, and Leydig cells could be labeled using removable plastic-embedded sections and immunogold-silver staining (IGSS). By using short periods of microwave irradiation for incubation of the monoclonal antibodies and the protein A-colloidal gold solution, the detection of BrdUrd-labeled cells could be remarkably enhanced. A comparative study of BrdUrd labeled spermatogonia in the testis of a Cpb-N mouse that received both [3H]-thymidine and BrdUrd proved that 90% of the BrdUrd-labeled cells also showed [3H]-thymidine labeling. The radioactive [3H]-thymidine labeling was a time-consuming method of 4 weeks' duration, whereas the BrdUrd-labeled cells could be labeled, fixed, enhanced, and counterstained in less than 3 hr. This investigation proves that BrdUrd labeling of S-phase cells can be a reliable, reproductive, rapid, and non-radioactive alternative method for [3H]-thymidine labeling of proliferating cells.
Sleep deprived rats undergo a predictable sequence of physiological changes, including changes in skin condition, increased energy expenditure, and altered thermoregulation. Amino-cupric-silver staining was used to identify sleep deprivation related changes in the brain. A significant increase in staining was observed in the supraoptic nucleus (SON) of the hypothalamus of rats with high sleep loss (>45 h) vs. their yoked controls. Follow-up experiments showed that staining was not significantly different in rats sleep deprived for less than 45 h, suggesting that injurious sleep deprivation-related processes occur above a threshold quantity of sleep loss. These anatomical changes suggest that the effects of sleep deprivation may be related to protein metabolism in certain brain regions.
A reduced silver technique using physical development to stain embryonic nervous tissue is described. Brains are fixed in Bodian's fixative. Paraffin sections are pretreated with 1% chromic acid or 5% formol. They are impregnated with 0.01% silver nitrate dissolved in 0.1 M boric acid/sodium tetraborate buffer of pH 8 or with silver proteinate. Finally they are developed in a special physical developer which contains 0.1% silver nitrate, 0.01-0.1% formol as reducing agent, 2.5% sodium carbonate to buffer the solution at pH 10.3, 0.1% ammonium nitrate to prevent precipitation of silver hydroxide, and 5% tungstosilicic acid as a protective colloid. The development takes several minutes in this solution, thus the intensity of staining can be controlled easily. The method yields uniform, complete and reproducible staining of axons at all developmental stages of the nervous tissue and is easy to handle.
We have developed a method to detect oligoclonal IgG bands in unconcentrated CSF from patients with MS or guinea pigs with chronic relapsing experimental allergic encephalomyelitis, by isoelectric focusing (IEF) in polyacrylamide gel and silver staining. Five to 10 microliters of CSF was sufficient. The bands were identified as IgG in immunofixation after IEF.
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The activity of nucleolar organizer regions (NORs) in chromosomes and interphase nuclei of bone marrow (BM) cells from 21 patients with chronic myelocytic leukemia (CML), including seven patients at the time of blastic crisis (BC), has been studied with silver nitrate staining. The average numbers of Ag-NOR per metaphase in PHA-stimulated peripheral blood lymphocytes from patients with CML and normal individuals were 7.1 +/- 0.3 and 7.4 +/- 0.1, respectively, indicating no statistical difference between them. Those in BM cells from patients with CML, as in the normal donors, were more heterogeneous compared to PHA-stimulated lymphocytes, and most of the metaphases (up to 67%) did not contain silver-stained NORs. The average number of Ag-positive NORs in BM mitoses from untreated patients in the chronic phases of CML and from those in the BC were similar (4.9 +/- 0.3 and 4.8 +/- 0.4, respectively). As for NORs of the Ph chromosome, they were Ag-positive in the majority of patients, including 9 of 14 in the chronic phase and 3 of 7 in the BC. This article contains some data in support of the authors' previous assumption regarding the correlation between BM Ag-NOR patterns and the degree of maturity of the cells tested in mitosis.
T and Tn are pancarcinoma epitopes (EPs) which can be immunodetected in about 90% of adenocarcinomas (CAs). To study the location of T and Tn EPs and their relations to adhesion plaques on CA cells, immunogold-silver staining method was employed at scanning electron microscope (SEM) level. Human breast CA cells grown on coverslips were fixed in paraformaldehyde and glutaraldehyde, reacted with cocktails of monoclonal antibodies against T or Tn EPs, followed by incubation with 10 nm gold conjugated goat anti-mouse immuno-globulins. The positive gold particle labelling was amplified with silver enhancement solution, and the specimens were then routinely critical point dried, sputter-coated with palladium and observed under SEM. The studies show that T and Tn EPs are not randomly distributed on CA cell surface; they are aggregated at the adhesion plaque area. These results confirm that T and Tn EPs play roles in CA cell adhesion, and suggest that they may represent the initial points of contact by immuno-effectors and therefore can be utilized in immunointervention and anti-adhesion therapy against CA.
A modified acetylcholinesterase-silver technique has been used to study the nerve terminal innervation in human muscle removed at biopsy and in post-mortem material. The results of "Functional Terminal Innervation Ratio's" are given, and the advantages of using this method are discussed.
A new method is described which allows light microscopy visualization of the synaptonemal complexes in primary spermatocytes. The conventional acetic acid-alcohol fixed and air-dried preparations can be used for the modified silver staining technique. Several hundred pachytene spermatocytes from a single mouse testis or human testicular biopsy can be examined. The prospective utilization of the present method is briefly discussed.
A multiplex PCR system has been developed to amplify 4 Y-chromosome specific short tandem repeats (STR), DYS391, GATA-A4, GATA-A10 and GATA-H4, simultaneously. PCR products were separated by polyacrylamide gels electrophoresis followed by silver stain. When 311 unrelated males from the Han population in Guangdong were detected by the multiplex system, DYS391, GATA-A4, GATA-A10 and GATA-H4 showed 5, 7, 6 and 5 alleles respectively. Total 98 haplotypes could be identified. Gene diversity value for the 4 STR was 0.4623, 0.6972, 0.7173 and 0.6015 respectively. The gene diversity value for the haplotypes of the 4 Y-STR reached 0.9755. The four Y-STR multiplex system will be very powerful for establishing Y-STR database, exploring human origin, paternity testing and personal identification.
Three enzyme-linked immunosorbent assays (ELISAs) (rotavirus enzyme immunoassay [EIA; International Diagnostic Laboratories], Pathfinder [Kallestad Laboratories], and Rotaclone [Cambridge Bioscience, Inc.]) and hybridization of viral RNA with a nonradioactive, synthetic oligonucleotide DNA probe (SNAP; Molecular Biosystems, Inc.) were compared with silver-stained polyacrylamide gel electrophoresis (PAGE) of viral RNA for the detection of rotavirus in fecal specimens. Hybridization was performed following column purification of the viral RNA. Of 286 specimens analyzed by PAGE, SNAP, rotavirus EIA, Pathfinder, and Rotaclone, 88 were positive by PAGE. All 88 specimens were also positive by the other four assays. Nine specimens that were positive by one or more of the assays were positive by blocking ELISAs but were negative by PAGE. If these nine specimens were considered to be true positives, the final sensitivities and specificities were as follows: PAGE, 91 and 100%; SNAP, 94 and 97%; rotavirus EIA, 96 and 97%; Pathfinder, 100 and 94%; and Rotaclone, 96 and 97%, respectively.
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A new double immunohistochemical technique for the simultaneous detection of B-cells and T-cells was investigated, using tissue preparations obtained from human axillary lymph nodes and rejected renal allografts. The specimens were immunostained first for the demonstration of B-cells, by the immunogold-silver staining (IGSS) method using Leu-12 monoclonal antibody, and then for T-cells by the avidin-biotin-peroxidase complex (ABC) method using Leu-1 monoclonal antibody. With the present methods, both B-cells and T-cells were clearly detected and distinctively identified without cross-linking of antibodies or double reaction of enzymes.
We have investigated the potential of two-dimensional electrophoresis for identifying differences in protein patterns between acute lymphocytic (ALL) and acute myelogenous leukemia (AML). We studied blood cells directly isolated from seven children with non-T, non-B ALL and five with AML. Solubilized proteins were electrophoresed with the ISO-DALT system and made visible by silver staining. More than 800 spots were routinely detected in individual gels. Patterns for multiple runs of the same sample were indistinguishable. Most of the prominent spots were present in gels from all 12 of the individuals studied. However, substantial variability in spot intensity was observed, even among patients in the same group. Five spots were consistently present in gels of all the AML patient, and either completely absent or substantially decreased in ALL gels. Three other spots were present in ALL but not AML gels. We conclude that this may be a useful tool for characterizing leukemic cells.