Fluor-o-4 Slide Set # 92000
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$220
This set of four fluorescently labeled slides is designed to act as a reference standard for microscope performance. In each case they are optimized for specific performance parameters of the instrument, including resolution, cube performance or spectral separation and camera sensitivity. All four slides are labeled with the same fluorophores: Hoechst (Sigma), Alexa 488 (Molecular Probes), Cy3 (Amersham) and Cy 5 (Amersham). These fluorophores were selected as they allow 4-color imaging with discrete excitation and emission spectra on most currently available microscopes. Regardless of whether you use these slides on a widefield or confocal microscope, you should be able to image all 4 colors with minimal bleed through between channels if the microscope is properly set up.
There are 4 separate tissue samples in each set: gut, liver, pancreas, and skin. The sections are mounted in a proprietary aqueous mountant. We chose to use an aqueous preparation to allow high resolution DIC imaging (for example, examine the brush border of the epithelial cells in the section of gut). This mountant is robust and can be considered a permanent mount designed to minimize photobleaching. However, as is the case with all fluorescently labeled samples, the dyes are sensitive to their environment, will quench under extended illumination, and are to some degree sensitive to temperature. These slides can be stored indefinitely at 4° C, and should last at least 6-8 months at room temperature. The goal behind this product is to generate a consistent and “real world” slide set. Importantly, these slides are labeled to highlight high abundance cellular constituents within the tissues, so if you are studying a low abundance or unknown protein you may not achieve equivalent labeling.
Typical widefield sets: 31000, 41001, 41007a, 41008. Confocal/laser sets vary by instrument.
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Liver Section
Nuclei: Hoechst dye
Peroxisomes: Alexa 488
SE1: Cy3 Actin: Cy5
The liver, after the skin, is the largest organ in the body. Its job is to process a huge variety of nutrients for use by individual cells throughout the body. The primary cell type within the liver is the hepatocyte. This section is one of our favorite preparations as a resolution standard and is in many ways the hallmark product of this slide set. The key labeling components of this slide are the peroxisomes. Each peroxisome is labeled with a primary antibody to PMP 70. This molecule is abundant, and essentially delineates the peroxisomes within the hepatocyte. The size of individual peroxisomes varies between about 200 and 400 nm in cross section and the label used here is specific to the surface of the organelle. As such they can be used as a resolution standard for light microscopy. If you examine the slide using a high numeric aperture (1.3 or better) objective, it should be possible to see the peroxisomes as “donuts” within the cell, at lower numeric apertures they appear as round solid structures. Since the resolution of any microscope has a linear relationship between numeric aperture in the XY plane and the square of NA in the Z axis, this sample becomes an ideal test for both widefield and confocal microscopes. When examining this slide with a confocal, you should be able to optically section the peroxisomes such that they appear as defined structures with a bright periphery. Notably the center of the peroxisome shows a clear intensity dip in confocal.
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Liver Section
Nuclei: Hoechst dye Peroxisomes: Alexa 488 SE1: Cy3 Actin: Cy5
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Skin
Nuclei: Hoechst dye
Actin: Alexa 488
Elastin: Cy3 Collagen: Cy5
The skin is the largest organ in the body, it cushions the body from assault, is the primary surface for heat regulation, and is a primary tactile or sensory surface. In this section the most obvious cellular structure is the stratum corneum. This is the surface of the skin, and constitutes the top ten or so cell layers visible in the section. Immediately beneath the epidermis is a thin though tightly defined layer of basement membrane, in this case labeled with antibodies to a primary constituent (collagen IV). This thin layer essentially separates the epidermis from the dermis. The dermis has a rich blood supply and constitutes the “elastic” part of the skin. We have labeled this component with antibodies to elastin. This tissue was chosen as it represents one of the more difficult structures in the body to image; apart from the principal cellular structures defined above you should also see significant autofluorescence from the elastin. This is particularly evident when using excitation from the UV through blue excitation. In fact it is reasonable to use autofluorescence to collect a useful image of the elastin in skin or large blood vessels. In this case, you should be able to selectively exclude the elastin autofluorescence when using a spectral confocal.
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Skin
Nuclei: Hoechst dye Actin: Alexa 488 Elastin: Cy3 Collagen: Cy5
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Gut
Nuclei: Hoechst dye
Tubulin: Alexa 488
Laminin: Cy3 Actin: Cy5
The ileum is one of the primary absorptive surfaces in the body. This slide is perhaps the most dramatic of the collection, and should produce the most graphically satisfying images. The apex of each epithelial cell is covered with actin rich microvilli, about 400 nm long that should be easily defined with any high numeric aperture objective above 40X. The goblet cells which intersperse the absorptive columnar epithelia can be seen to be full of mucus droplets. This is perhaps best seen using differential interference contrast. Under the epithelium, is the lamina propria which is highly vascularized.
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 Gut
Nuclei: Hoechst dye Tubulin: Alexa 488 Laminin: Cy3 Actin: Cy5
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Pancreas Nuclei: Hoechst dye
Actin: Alexa 488
Insulin: Cy3 Laminin: Cy5
This is the largest single section in the collection. The individual pancreatic islets are stained red to highlight the beta cells. Importantly these structures, like the peroxisomes in the liver sample, should be visible as discrete structures at high resolution. However, unlike the peroxisomes, the beta cell granules are stained throughout and do not appear hollow.
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Pancreas
Nuclei: Hoechst dye Actin: Alexa 48 Insulin: Cy3 Laminin: Cy5
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