Journal of Histochemistry & Cytochemistry

Chen, K; Wight, T N

doi: 10.1177/32.4.6200530pmid: 6200530

The extracellular matrix in cultures of arterial smooth muscle cells has been examined by ultrastructural histochemistry using each of the following cationic dyes: ruthenium red, Alcian blue, acridine orange, and safranin O. All dyes exhibited an affinity for a structural component that was either preserved as a granule with ruthenium red or Alcian blue, or as an extended filament or bottlebrush structure with acridine orange or safranin O. Both granules and filaments were removed when the cultures were pretreated with chondroitinase ABC, an enzyme that degrades the glycosaminoglycan moiety of some proteoglycans. These structural components of the extracellular matrix were not observed when cultures were prepared in the absence of the cationic dyes. Labeling experiments (35S-sulfate) revealed that approximately 40% of the total labeled proteoglycans were lost during routine processing for electron microscopy (i.e., fixation through dehydration). Inclusion of any one of the cationic dyes during fixation reduced the losses to less than 1%. The extended filamentous structure preserved by safranin O and acridine orange resembled the structure of purified proteoglycans prepared from the same cultures and spread on cytochrome c monolayer films. These observations suggest that proteoglycans exist as extended bottlebrush structures within the extracellular matrix, and support the interpretation that the granular deposits observed in the ruthenium red and Alcian blue preparations most likely represent individual proteoglycan monomers that have undergone molecular collapse during processing. In addition, the dyes also exhibited an affinity for chords of fine fibrils that contained small granules and/or filaments. Both the fibrillar material and the associated granular and filamentous structures enmeshed in the fibrils resisted digestion with chondroitinase ABC.

Paradis, I L; Merrall, E J; Krell, J M; Dauber, J H; Rogers, R M; Rabin, B S

doi: 10.1177/32.4.6368677pmid: 6368677

The reliability and sensitivity of an indirect avidin-biotin-peroxidase (ABC) procedure for enumerating lymphocyte subpopulations was compared to flow cytometry (FC) employing direct immunofluorescence. Lymphocytes were enumerated by two different methods. For counting method I, which is the method of conventional FC, the number of immunostained lymphocytes was compared to the total number of lymphocytes present. The ABC procedure by method I detected a greater proportion of immunostained lymphocytes for all subsets tested than did FC. By counting method II, where the number of immunostained lymphocytes is compared to the total number of cells present, the ABC analysis still detected more total T cells than FC but the results for the two analyses were similar for T helper and T suppressor cells. Thus, the ABC technique appears to be a valid method for enumerating T lymphocyte subsets. Furthermore, as compared to FC, it offers the advantages of reduced cost, simplicity of understanding and performance, need for fewer cells, and a permanent record of lymphocyte staining. For these reasons, we feel that the ABC technique will enjoy widespread application for the identification of lymphocyte membrane antigens.

Tunkel, A R; Studzinski, G P

doi: 10.1177/32.4.6608553pmid: 6608553

The aminonucleotide of puromycin (AMS) is known to have a differential effect on ribosomal RNA (rRNA) synthesis and the cell-cycle traverse in normal as compared to neoplastic or virally transformed cells. In this study, AMS and its demethylated derivative, 3'-amino-3'-deoxyadenosine (3'-AmA) have been used to compare their effects on normal (IMR 90) versus transformed (AG 2804) human fibroblasts with regard to preribosomal RNA (pre-rRNA) transcription, the processing of this RNA, and structural changes in the nucleolus. The processing of pre-rRNA in normal and transformed fibroblasts treated with 3'-AmA for 4 hr was markedly depressed. However, this process did not appear to be affected by the AMS treatment of normal cells, while in transformed cells it was maximally inhibited within 4 hr of exposure to this drug. Ultrastructural changes were observed in the nucleoli of normal and transformed cells treated with 3'-AmA, whereas treatment of these cells with AMS produced alterations of nucleolar structure only in the transformed cells. These changes correlated with the onset of inhibition of pre-rRNA processing. Our findings suggest that the impairment of pre-rRNA processing produced by AMS and 3'-AmA in transformed cells and by 3'-AmA in normal cells may be due to structural disorganization of the nucleolus produced by these antimetabolites.

Evans, N R; Webb, H E

doi: 10.1177/32.4.6323573pmid: 6323573

Processing tissue for transmission electron microscopy by standard laboratory methods can take two to three days. This makes the development of new techniques time consuming and generally restricts the use of the electron microscope in routine diagnostic work. The possibility of viewing tissue with the electron microscope five hours after sampling using rapid processing techniques is presented. The morphology of the tissue appears undamaged with cell and organelle ultrastructures being readily recognized, as is the presence of virus and its replicating stages. When combined with immunoelectron microscopy a rapid labeling protocol is possible. We have used the technique to develop protein A-gold (6 and 16 nm particles) and ferritin immunoelectron microscopic techniques to demonstrate viral antigens in brain cell cultures and brain tissue from mice infected with Semliki Forest virus.

Hall, J; Talamantes, F

doi: 10.1177/32.4.6368678pmid: 6368678

The localization of mouse placental lactogen (mPL) in the mouse placenta has been examined with the avidin-biotin-peroxidase complex (ABC) technique. Staining was observed to occur in the basal zone of placentae from days 10, 12, 15, and 18 of gestation. Within the basal zone, the most prominent staining was localized to the cytoplasm of the giant cells. Within the labyrinth, staining was observed in the giant cells in day-15 and day-18 placentae, with a greater number of stained cells being observed in the latter.

Ahumada, G G; Saffitz, J E

doi: 10.1177/32.4.6707462pmid: 6707462

Fibronectin, a glycoprotein that binds to collagen and modifies the adhesion properties and motility of cells in culture, is present in the interstitium of rat hearts. To localize fibronectin more precisely and to assess its relationship to the myocyte and to connective tissue elements, we employed a double antibody technique to label myocardial fibronectin with electron-dense ferritin to permit an ultrastructural analysis. Fibronectin was found to be associated with collagen, and in some cases appeared to link collagen fibers. Fibronectin was also found inserted along the surfaces of cardiac myocytes, connecting these cells to perimyocytic collagen. These ultrastructural relationships imply that fibronectin is a major component of the myocardial interstitium, and may affect myocardial compliance and control the motion of myocytes during the contraction and relaxation of the heart.

Kramps, J A; van der Valk, P; van der Sandt, M M; Lindeman, J; Meijer, C J

doi: 10.1177/32.4.6561228pmid: 6561228

The immunohistochemical results obtained with antibodies directed against human neutrophil elastase is described. It was found that neutrophil elastase can be used as a specific marker of cells of the neutrophilic lineage. In normal hematopoietic cell preparations, only promyelocytes and more differentiated myeloid cells stain positive for elastase. In acute or chronic myeloid and myelomonocytic leukemia, the same neutrophil myeloid cells stain positive, whereas neoplastic cells of the monocytoid line are negative. Using elastase in conjunction with other markers, it is possible to differentiate easily the involvement of different cell lines in myeloproliferative diseases.

Houser, C R; Barber, R P; Crawford, G D; Matthews, D A; Phelps, P E; Salvaterra, P M; Vaughn, J E

doi: 10.1177/32.4.6368679pmid: 6368679

Spurious staining related to the second (linking) antibodies was observed in immunocytochemical specimens processed with an unlabeled antibody method. Some of this staining was suspected to result from species cross-reactivity of the second antibodies with endogenous immunoglobulin Gs in the tissue. Therefore, species-specific second antibodies were obtained, and the staining patterns of tissue processed with such antibodies were compared with those of tissue processed with standard (nonspecies-specific) second antibodies. In these studies, a monoclonal antibody to choline acetyltransferase (ChAT) was utilized as the primary antibody, and a similarly prepared monoclonal antibody that did not react with ChAT served as a control antibody. Spurious staining that included staining of discrete tissue and cellular components as well as amorphous background staining was present in both control and experimental tissue processed with standard second antibodies. Such staining was virtually eliminated in tissue processed with species-specific second antibodies. In specimens from the central nervous system, for example, species-specific second antibodies greatly reduced dark staining within the area postrema, in the pia-arachnoid membranes, and around blood vessels as well as the staining of small dot-like structures within some large neurons. In addition, the general level of background staining was reduced in both adult and developing tissues, thus permitting clearer visualization of many positively stained structures. In peripheral tissues such as skeletal muscle, spurious staining of connective tissue elements was eliminated, allowing the observation of previously occluded ChAT-positive structures such as nerve fibers and motor end-plates. Thus, species-specific second antibodies appear to be very useful for immunocytochemistry, particularly when the primary antibody and the tissue to be studied are from closely related species.

Hand, A R; Oliver, C

doi: 10.1177/32.4.6142913pmid: 6142913

The structure and cytochemistry of the Golgi apparatus and GERL of rat parotid acinar cells was studied after in vivo secretory stimulation with isoproterenol. Discharge of mature secretory granules was complete within 1 hr after isoproterenol injection, but immature granules in the Golgi region or near the lumen were not released. At early times (1-5 hr) after isoproterenol, acid phosphatase (AcPase) activity was markedly increased in GERL and immature secretory granules compared to uninjected controls. GERL appeared increased in extent and numerous continuities with immature granules were observed. Reaccumulation of mature secretory granules was first evident at 5 hr, and was almost complete by 16 hr after isoproterenol. Thiamine pyrophosphatase (TPPase) activity, normally restricted to the trans Golgi saccules, was frequently present in immature granules during this time. Narrow cisternae resembling GERL, occasionally in continuity with immature granules, also contained TPPase reaction product. By 16-24 hr after stimulation, the activity and distribution of AcPase and TPPase were similar to control cells. These results demonstrate the dynamic nature of the Golgi apparatus and GERL in parotid acinar cells, and emphasize the close structural and functional relationship between these two structures.

Sklarew, R J

doi: 10.1177/32.4.6707463pmid: 6707463

A scheme has been developed for deciphering the cell cycle time parameters of cell subpopulations that differ in their DNA ploidy level and that coexist in mixed heteroploid tumors. The S-phase analysis is presented. The approach is coupled to an automated imaging methodology for simultaneous determination of the Feulgen-stained DNA content and grain count of 3H-thymidine-labeled cells in autoradiographs (Sklarew RJ: J Histochem Cytochem 30:35, 30:49, 1982). The experimental designs involve 3H- and 14C-thymidine double labeling and Colcemid incubation. The deconvolution of the S-phase ploidy composition is illustrated in rat sarcoma cultures comprising four major ploidy subpopulations; with G-2 and mitotic DNA contents of approximately 4C, 8C, 16C, and 32C. The components were identified by their DNA ploidy level, and their S frequencies and labeling indices were obtained. A scheme is also developed and validated for obtaining the S-phase emptying profile of component ploidy subpopulations, and their cell flux at the S/G-2 and G-2/mitosis phase boundaries. In the sarcoma cultures S mobility was found to decrease with increasing DNA ploidy level over the entire ploidy range.