RAPID COMMUNICATION |
Correspondence to: F. Susan Wong, Section of Immunobiology, Yale U. School of Medicine, 310 Cedar Street, New Haven, CT 06520..
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Summary |
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We developed a simple method employing the use of flat-embedding techniques on thick frozen sections which allows correlation of light and electron microscopic immunohistochemistry. This method has been particularly useful in visualization of pancreas sections, an adaptation especially important because this tissue is not amenable to conventional vibratome sectioning. In this study we demonstrate the use of this technique to examine the same tissue section at the light and the electron microscopic level while maintaining morphology. (J Histochem Cytochem 46:13411345, 1998)
Key Words: immunohistochemistry, frozen section, correlative light and electron microscopy, CD8 T-cells, pancreas
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Introduction |
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Insulin-dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disease that results in the destruction of insulin-producing ß-cells in the pancreatic islets of Langerhans. To better understand the mechanism of pathogenesis and the roles of cytotoxic subsets of cells, visualization of the morphological detail of cellular and subcellular components involved in pancreatic destruction at the ultrastructural level is important. Therefore, we have developed a simple method for concurrent visualization of immunohistochemistry of light and electron microscopic material for soft or fatty tissue.
Until now, most electron microscopic (EM) techniques for visualizing the pancreas have utilized cells and islet co-culture, followed by pre-embedding immunohistochemistry (IHC) and thin sectioning of cell pellets (
Flat-embedding techniques were originally developed for Golgi electron microscopy (
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Materials and Methods |
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Tissue Preparation
Adoptive transfer experiments were performed in which 68-week-old NOD/Caj mice were irradiated and CD8 islet-specific cloned T-cells bearing a T-cell receptor (TCR) using the Vß6 gene segment were transferred into the mice IV as described previously (
Immunohistochemistry
Thick (2025-µm) sections were cut in a cryostat and affixed to Thermanox plastic coverslips (EMS). Immunostaining was performed as previously described (
Tissue Preparation for EM
The sections were then washed, postosmicated (1% OsO4 in 0.1 M PB for 15 min), dehydrated through increasing concentrations of ethanol (the 70% ETOH contained 1% uranyl acetate), cleared in propylene oxide (PO), and placed in a mixture of POEpon 812, 50 /50 v/v (EMS). Coverslips were again placed on glass slides for microscopic visualization of islets that were to be ultrasectioned for electron microscopy. Once these areas were identified, as in Figure 2A, the sections were first covered with a drop of 100% Epon 812 and then a gelatin capsule filled with Epon 812 was positioned over the areas of interest. Usually more than one capsule was placed on the section. After polymerization in a 60C oven for 48 hr, the coverslips were removed and the blocks photographed as in Figure 1A for further correlation. Ultrathin sections were cut on a ReichertJung Ultracut E (Leica; Deerfield, IL). Grids were observed in a Philips CM10 electron microscope. Further contrasting with lead citrate was unnecessary.
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Results |
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The results demonstrated that this method allows the correlation of both surface and intracellular stain in the sections under light and electron microscopy. The CD8 coreceptor is a cell surface marker. At the light microscopic level, the immunoperoxidase reaction product was ringed in appearance and was associated with the cell membrane of the T-cells (Figure 1A). Correlative EM examination confirmed that the staining of the CD8 co-receptor is indeed specific for the surface membrane of the cloned T-cells and is not found on the pancreatic ß-cells (Figure 1B). Pseudopodia from the T-cells were routinely seen extending into the ß-cells in the late phases of cytotoxicity (Figure 1B).
In contrast, insulin is an intracellular marker that is present in the secretory granules of pancreatic ß-cells. The immunoreaction for insulin at the LM level appeared as patchy cytoplasmic staining (Figure 2A). Electron microscopic observations of the same tissue showed the subcellular localization of insulin to be specific for the secretory granules (Figure 2B).
The ultrastructural preservation was excellent in all of the preparations, as evidenced by completely intact intracellular organelles such as endoplasmic reticulum, lysosomes, and mitochondria, and by undamaged cellular and nuclear membranes (Figure 1 and Figure 2). The tissue antigenicity was also well preserved, which is clearly demonstrated by the highly specific and intense immunoreaction for both antibodies and particularly for the cell surface marker.
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Discussion |
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This method is an adaptation of standard techniques that allows examinination of the same frozen section at the light and electron microscopic levels. This is important because it enables investigators to examine soft tissues, such as the pancreas, in the natural history of disease or after manipulations. The ability to look at cellular interactions and changes in surface and intracellular markers is very useful in the investigation of pathogenic processes.
Essentially, the conditions of tissue preparation are those used for light microscopy, which reduces the time required at the initial stages. Although the frozen sections are thick, this technique allows clear visualization of immunostaining and the ability to verify the specificity of the antibody reaction before postosmication and embedding for EM. In addition, LM photographs can be taken either before or after EM polymerization, and thus the immunohistochemical results can be correlated at the light and electron microscopic levels for both membrane and intracellular staining. Furthermore, the ultrastructural preservation of the tissue is maintained. Fixation of tissue before immunostaining helps retain antigens and preserve morphology. Sucrose infusion and embedding of tissue in an isopentane bath on dry ice aids in reduction of empty spaces around cells caused by intracellular ice crystal formation.
An alternative procedure to use of the vibratome for thick sections is the sectioning of free-floating frozen tissue with a freezing microtome or cryostat (
Major advantages of the use of Thermanox plastic cover slips in this method are that the tissue adheres throughout the entire procedure, there is good visualization by light microscopy, and the tissue is flat-embedded on the coverslip itself. After polymerization the coverslip can be easily removed. A similar approach has been used by
We believe that the modifications that we have made in this study advance our ability to correlate immunohistochemistry at the LM and EM level while maintaining good morphology and definition of ultrastructure at the EM level. This approach will be generally important in the analysis of cellular interactions and the localization of surface and intracellular antigens after immunostaining in soft tissue.
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Acknowledgments |
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Supported by a Career Development Award from the Juvenile Diabetes Foundation International to FSW and the National Institutes of Health grant PO1-DK53015 to CAJ. CAJ is an investigator of the Howard Hughes Medical Institute.
We gratefully acknowledge the assistance of Hannah Janeway in the preparation of this manuscript.
Received for publication August 25, 1998; accepted August 25, 1998.
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