Biology Reference
In-Depth Information
4. Quality differences between paraffi n- and CMC-embedded
samples were shown in Bioanalyzer surveys. In these experi-
ments conventional paraffi n-embedded samples displayed a
higher degree of RNA degradation compared to the CMC-
embedded specimens [
17
].
5. In a comparative study, paraffi n ribbons were exposed to water
for different time periods with and without RNase inhibitor
and drying temperatures of 42 and 4 °C. It was demonstrated
that avoiding the 42 °C drying step resulted in decent RNA
quality and the RNase inhibitor was most effi cient in the fi rst
hour. Even better results were obtained after removing the
water and drying the specimens at 4 °C for at least 1 h [
16
].
A different way to avoid the water and drying step is using the
paraffi n-tape transfer system [
10
]. The system is based on
adhesive tapes to capture sections instead of using a brush for
transferring them to the slides, which avoids rolling or
breaking.
6. Prepare fresh before use.
7. Place paraffi n chips at 58 °C a day before use.
8. A wide variety of membrane slides are provided by different
suppliers. Typically they are coated with PEN: polyethylene
naphthalate (Zeiss, Leica, Arcturus); PET: polyethylene tere-
phthalate (Zeiss, Leica); PPS: polyphenylene sulfi de (Leica);
POL: polyester (Leica); FLUO: fl uocarbon (Leica).
9. 1× PBS (1,000 ml): 8 g NaCl, 0.2 g KCl, 1.44 g Na
2
HPO
4
,
0.24 g KH
2
PO
4
; adjust to pH 7.4.
10. A partial vacuum (200-400 mbar for 15-20 min) facilitates
the fi xative to penetrate the tissue [
2
,
8
,
15
,
16
].
11. Plant cells in contrast to mammalian cells have big vacuoles for
water storage. This is problematic for cryo-sectioning, because
during the fast freezing process ice crystals can form and
destroy the cellular structure. This can be avoided by treating
the plant tissue in sucrose solution.
12. Vacuum infi ltration (200-400 mbar on ice for 15 min)
facilitates the penetration of the solution into the tissue.
Acknowledgment
Root research in the laboratory of FH is supported by the DFG
(Deutsche Forschungsgemeinschaft) and the 7th Framework
Programme of the European Union.
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