Confocal microscopy and immunofluorescence protocols, image deconvolution, optimization methods and techniques for the research scientist.
Protocols: immunofluorescence, cryostat sections, sample preparation ...
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Cryostat tissue sections are often chosen for immunofluorescence procedures. Although frozen tissue sections are fragile, they exhibit favorable signal to noise characteristics and can yield good confocal microscopy images.
1. After excision, immerse the tissue sample in isobutanol (cooled to
-20 deg C) (i.e. use a 50 ml cold resistant tube with isobutanol for
a 1-2 g sample)
2. Carefully immerse the tube in liquid nitrogen to quickly deep freeze
the sample
3. Store at -80 C
4. In order to cryosection the sample, remove from -80 C storage, and
allow to equilibrate with the warmer cryostat temperature (-20 to -30
C)
5. Choose a section thickness (10 to 14 microns is good for confocal
microscopy)
6. Center the cut sections on poly-l-lysine
coated microscope slides.
7. Place the slides in a slide rack and store at -80 C or continue with
immunofluorescence.