Laser Microdissection of Plant Cells - Plant Cell Morphogenesis: Methods and Protocols - page 244

2. The plant tissue is dehydrated by an ethanol series (70, 80, 90,
and 100 %), followed by an ethanol:xylene or ethanol:isopropanol
treatment (3-4 h (v/v) 75 %:25 %; 50 %:50 %; 25 %:75 %;
100 %) at room temperature [ 7 , 17 , 18 ].

3. The samples are slowly embedded directly into paraffi n by a
gradient, starting with a 3:1 ratio of dehydration solution and
paraffi n for 3-4 h at 58 °C.
4. The dehydration solution:paraffi n ratio is changed to 1:1 and
the samples are incubated twice in this solution prior to
replacement by 100 % paraffi n [ 7 , 14 , 19 , 20 ]. Each step of
this sequence has an incubation time of 3-4 h at 58 °C.
5. The paraffi n-embedded samples can be stored at 4 °C prior to
downstream analyses.

4 Fixation and Embedding for Paraffi n Sectioning via Microwave Oven Technique

1. An alternative way of embedding plant samples in paraffi n is
the microwave oven technique. In this approach the samples
are fi xed by Farmer's solution in a specifi c microwave oven
(e.g., PELCO BioWave), for 15 min at 37 °C.
2. Farmer's solution is replaced by an ethanol series (50, 70, and
100 %) at 67 °C for 75 s per step.
3. Ethanol is replaced by a 50:50 ethanol:isopropanol mixture
for 90 s.
4. Samples are incubated in pure isopropanol at 77 °C for 90 s.
5. Isopropanol is replaced by a 1:1 isopropanol:paraffi n mixture
at 77 °C for 10 min followed by at least four exchanges of
paraffi n at 67 °C for 30 min each step [ 21 ].
6. Several modifi cations of this basic protocol have been applied
to different plant tissues [ 13 , 15 , 22 ].

5 Fixation and Embedding for Cryo-sectioning

1. Plant tissue for cryo-sectioning is fi xed in Farmer's solution
(see above for paraffi n sections) [ 2 , 3 , 8 , 11 ]. Alternatively, no
fi xation is applied [ 23 - 25 , 27 ].
2. Samples are incubated in 10 % sucrose solution (10 % sucrose
(w/v) in PBS) ( see Note 11 ) after fi xation at 4 °C for 1 h
( see Note 12 ).
3. The 10 % sucrose solution is replaced by 15 % sucrose (w/v)
in PBS at 4 °C between 1 and 24 h.