Immunochemistry on ultrathin frozen sections

Summary Ultrathin frozen sections can be cut smoothly from many fixed and appropriately treated specimens. To use such sections for immunochemical localization of intracellular antigens, fixa ion conditions must be selected to optimize at least three variables, namely, preservation of ultrastructure, preservation of antigenicity and retention of accessibility of the antigen to the antibody. Furthermore, staining of the sections must be such that both the immunolabels and structures are clearly recognized. Our efforts to attain these goals are described in relation to their historical background. Although there are still problems to be solved and improvements to be made, we now consider that cryoultramicrotomy has reached the stage of being useful in studying many questions which will not be easily approached otherwise.This article is dedicated to the memory of Dr Wilhelm Bernhard, Institut de Recherches Scientifique sur le Cancer, Villejuif, France.     

Culture and microscopy of microorganisms in frozen sections.

A simple method is described in which frozen sections are prepared from tissue cell suspensions with added microorganisms and then cultured. The enhanced growth of the organisms makes their indentification easier. A number of applications is suggested.     

Intracellular localization of certain membrane glycoproteins in mouse T-lymphoma cells using immunoferritin staining of ultrathin frozen sections

Comparative studies on the cellular morphology of culturd mouse T-lymphoma cells (with particular emphasis on organelles and membrane-associated materials) were conducted using both frozen thin sections and epon thin sections. Due to the fact that the frozen thin sectioning technique allows antigenicity to be retained and also permits good accessibility of the external macromolecular reagents to the interior of the cell, we have been able to explore the intracellular localization of some membrane glycoproteins such as Con A-binding sites and viral membrane glycoprotein, gp 69/71. Our data indicate that most of the membranous cellular structures (e.g., rough endoplasmic reticulum, vesicles, Golgi and nuclear envelope) contain the Con A-specific sugars, mannose, and glucose. In addition, we have found that intracellular gp 69/71 molecules exist in an aggregated form at the terminal region of cisternae of rough endoplasmic reticulum and in vesicles of two size ranges (0.1 to 0.15 microns and 0.3 to 0.4 microns) as well as in the cytoplasm close to the plasma membrane. These findings have not only confirmed some of the previous biochemical data but have also provided new information concerning the biochemical nature of intracellular membrane components and the possible biosynthetic fate of membrane precursor molecules.     

Immunoelectronmicroscopy of neural antigens on ultrathin frozen sections

We have utilized immunocryoultramicrotomy to detect synapsin, somatostatin, parvalbumin, and tubulin at the ultrastructural level. Immunocryoultramicrotomy combines informative identification of morphology with accurate immunolabeling. Moreover, since no detergents or organic solvents are used to enable antibody penetration, and since no enzyme marker diffusion occurs, localization of the antigens should be more accurate. Accordingly, it was possible to localize precisely all four antigens within a well-preserved structure. Application of this method has important advantages for high-resolution localization of molecules relevant to neuronal function.     

In situ visualization of hemopoietic cell subsets and stromal elements in rat and mouse bone marrow by immunostaining of frozen sections

We have developed a method to section frozen long bones of rat and mouse and stained bone marrow (BM) by (double) immunofluorescence and immunoperoxidase. Here we report this method and reveal the location of early hemopoietic progenitors (Thy-1) and myeloid cells (Mac-1) in mouse BM, and early hemopoietic progenitors and lymphoid cells (Thy-1), erythroid cells (HIS49), and macrophages (ED2) in rat BM. In mouse BM our new findings include (a) the scattered localization of early hemopoietic progenitors (Thy-1low) all over the marrow, and (b) the presence of Thy-1+ stromal cells, mainly subendosteally. In rat BM an important finding is that of (a) a subendosteal region of 12-14 hemopoietic cell layers characterized by an abundance of Thy-1 and the virtual absence of erythroid cells, and (b) the scattering of Thy-1very bright cells which are candidates for the earliest hemopoietic progenitors in this species. The results illustrate that the technique is an excellent tool for studying the topology of BM as an organ of hemopoiesis.