Mounting Live Cells onto Microscope Slides
Adapted from Imaging: A Laboratory Manual (ed. Yuste). CSHL Press, Cold Spring Harbor, NY, USA, 2010.INTRODUCTION
This protocol describes the mounting of cells which have been grown on cleaned coverslips in cell culture dishes containing the appropriate medium and supplements. Labeling is performed with the coverslips placed in culture dishes. After cells are labeled, the coverslips can be mounted onto microscope slides to be viewed for short-term observation of live cells or longer-term observation of fixed cells. For viewing live cells for longer periods of time, specialized chambers should be used in which suitable growth conditions can be maintained.
MATERIALS
Reagents
Cells of interest, grown on coverslips and labeled with fluorescent probe(s)
Phosphate-buffered saline (PBS)
Prepare PBS with added CaCl2 and MgCl2 (PBS+). This solution allows cells to adhere to each other and to the substrate. If cells are in medium containing no Ca2+ or Mg2+, they will round up and detach from the substrate.
Hanks’ balanced salt solution (HBSS) with Ca2+ and Mg2+ but no phenol red (Invitrogen 14025-092) can be used instead of PBS+ (see Step 1 note).
The wax mixture should spread smoothly and dry quickly on a glass slide. If it hardens too quickly, add more Vaseline and lanolin; if it does not harden quickly enough, add more paraffin.
Equipment
Cell culture dishes, sterile
Cotton swabs
Hot plate
Use to maintain Valap at the proper temperature.
Incubator, water-jacketed and CO2-regulated
Microscope, fluorescence
Microscope slides, cleaned
Alternatively, commercially available specialized chambers can be used on which to mount coverslips for imaging.
Parafilm
Tweezers, fine
METHOD
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1. Using fine tweezers, transfer live cells that have been grown on coverslips and labeled to a dish of PBS+ (or the desired cell culture medium).
Some cell culture media supplements, such as phenol red, can interfere with fluorescence observation. PBS+ or HBSS with Ca2+ and Mg2+ but no phenol red are frequently used when observing fluorescence to reduce or eliminate this problem. If cells are fixed before mounting, they can be mounted in commercially available aqueous mounting medium.
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2. Place narrow strips of Parafilm onto a microscope slide, positioned so that the coverslip will fit between them with two parallel edges of the coverslip extending onto the Parafilm strips.
The Parafilm serves as a spacer, which prevents live cells from being crushed. Cells that are fixed before mounting do not require spacers.
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3. Transfer the coverslip to the prepared slide.
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4. Using a cotton swab, seal the coverslip around its edges with melted Valap.
Avoid using commercial slide sealants that contain organic solvents (e.g., acetone), because they can be detrimental to live cells.
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5. Image the cells using fluorescence microscopy.
See Troubleshooting.
TROUBLESHOOTING
Problem: Images are blurred, distorted, or lacking in contrast.
[Step 5]
Solution: Poor-quality images can result from using plastic. This is particularly a problem when phase-contrast or differential interference contrast microscopy is used. It can also be a problem for transmitted light images on confocal microscopes. Some plastics interfere with fluorescence only at certain wavelengths. To avoid this problem, grow cells on optical-quality glass; #1.5 microscope coverslips are optimal for most uses. There are also optical-quality cell culture dishes available for situations when coverslips and slides are not feasible.
ACKNOWLEDGMENTS
I thank my wife, Nancy, and my daughter, Bryanna, for their patience while I was writing this article. I dedicate this article in memory of my mother, Cozette Chazotte, 1919-2009.
