In Situ Embedding of Cell Monolayers on Untreated Glass Surfaces for Vertical and Horizontal Ultramicrotomy.

Author(s):  
Kai Chien

Untreated glass is the ideal supporting substrate for cell cul¬ture growth since it is extremely flat and smooth, transparent and insoluable in organic solvent. However, difficulties have been encountered in removing polymerized epoxy resin from a glass surface following in situ cell monolayer embedment. Vari¬ous techniques have been made to grow cell cultures on either coated glass surfaces or plastic substrates. The purpose of this abstract is to describe a heat separation technique which when used together with a newly designed embedding mold allows cell monolayers to be transferred from untreated coverglass or glass slide to pre-shaped tissue blocks. The resulting tissue block can be easily separated and used directly for orientation light microscopy prior to ultramicrotomy.

Author(s):  
K. Chien ◽  
R.L. Van De Velde ◽  
R.C. Heusser ◽  
J.W. Said

In situ embedding is a preferable means for the ultrastructural study of cultured cell monolayers. However, the major difficulty is the separation of the polymerized plastic resin from the supporting substrates. This abstract demonstrates a simple, fast procedure for using existing products for cell culture and in situ embedding of monolayers. Epoxy block surfaces up to 10 cm2 can be easily separated from an untreated glass slide and sectioned vertically or horizontally for EM studies.The Lab-Tek tissue culture Chamber/Slides or Flaskette are constructed so that 1 to 8 chamber compartments or a single flask is attached to a regular glass slide by gaskets. Different cell lines or procedures can be performed on the same chamber/slide. When the plastic chambers and gasket are removed, the glass slide can be coverslipped and filed for reference. We use these products routinely for cell cultures. When an EM study is needed, we fix the cell monolayers in situ within the chambers or flask, dehydrate with ethanol, infiltrate and embed in an epoxy resin.


1991 ◽  
Vol 66 (5) ◽  
pp. 269-272 ◽  
Author(s):  
A. Beatrice Murray ◽  
Helga Schulze ◽  
Elisabeth Blauw

Author(s):  
Joseph E. Mazurkiewicz

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.


Author(s):  
E. S. Boatman ◽  
G. E. Kenny

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.


Author(s):  
K. Chien ◽  
R. Van de Velde ◽  
I.P. Shintaku ◽  
A.F. Sassoon

Immunoelectron microscopy of neoplastic lymphoma cells is valuable for precise localization of surface antigens and identification of cell types. We have developed a new approach in which the immunohistochemical staining can be evaluated prior to embedding for EM and desired area subsequently selected for ultrathin sectioning.A freshly prepared lymphoma cell suspension is spun onto polylysine hydrobromide- coated glass slides by cytocentrifugation and immediately fixed without air drying in polylysine paraformaldehyde (PLP) fixative. After rinsing in PBS, slides are stained by a 3-step immunoperoxidase method. Cell monolayer is then fixed in buffered 3% glutaraldehyde prior to DAB reaction. After the DAB reaction step, wet monolayers can be examined under LM for presence of brown reaction product and selected monolayers then processed by routine methods for EM and embedded with the Chien Re-embedding Mold. After the polymerization, the epoxy blocks are easily separated from the glass slides by heatingon a 100°C hot plate for 20 seconds.


Author(s):  
Amy A. Sutton ◽  
Clayton W. Molter ◽  
Ali Amini ◽  
Johanan Idicula ◽  
Max Furman ◽  
...  

Nanoscale ◽  
2017 ◽  
Vol 9 (36) ◽  
pp. 13820-13827 ◽  
Author(s):  
Sayantan Chatterjee ◽  
Uday Maitra

We describe a practical, one step, room temperature in situ synthesis of luminescent CdSe QDs using a bile salt derived metallogel, followed by their isolation from the gel and re-dispersion in an organic solvent.


Botany ◽  
2009 ◽  
Vol 87 (2) ◽  
pp. 210-221 ◽  
Author(s):  
Julia Nowak ◽  
Adam Nowak ◽  
Usher Posluszny

Compound palm leaf development is unique and consists of two processes. First, the primordial tissue folds through differential growth, forming plications. Second, these plications separate through an abscission-like process, forming leaflets. The second process of leaflet separation allows for the development of compound leaves. The question that this study addresses concerns the development of bifid leaves, as they do not form leaflets but only develop a cleft through an apical incision. The ideal genus to use for this study is Chamaedorea as it includes species with both pinnate and bifid leaves. Chamaedorea fragrans (Ruiz & Pav.) Mart. and Chamaedorea stolonifera H. Wendl. ex Hook. f. were chosen as the species with adult bifid leaves. Although Chamaedorea seifrizii Burret is a pinnate-leaved palm, its juvenile leaves are bifid. Scanning electron microscopy and light microscopy were used to study the development of bifid leaves. Our results indicate that neither of these bifid palms develop separation sites within the lamina, but rather the apical cleft develops through “late leaflet separation” or by an abscission-like process. In contrast, C. seifrizii juvenile leaves exhibit “early leaflet separation” when developing the apical cleft.


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