Biology Reference
In-Depth Information
was used [
3
,
11
]. Alternatively, no fi xation or embedding chemicals
were employed to avoid polymer peaks during the mass spectro-
metric identifi cation of peptides [
12
].
Table
1
summarizes the major characteristics of recently
performed plant LMD studies including the analyzed plant species,
cell type, the used LMD system, fi xation, tissue-embedding approach,
RNA extraction, amplifi cation step, and downstream analysis.
2
Materials
1. Farmer's fi xative: 3:1 ethanol: acetic acid (
see
Note 6
).
2. Ethanol series: 70, 80, 90, and 100 % (v/v).
3. Ethanol/xylene or ethanol/isopropanol (dehydration solution):
75 %:25 %; 50 %:50 %; 25 %:75 %; 100 % (v/v).
2.1 Paraffi n
Embedding
4. Melted paraffi n chips (
see
Note 7
).
5. Membrane slides (
see
Note 8
) or plain slides.
6. 100 % Xylene.
2.2 Microwave
Technique
1. Farmer's fi xative: 3:1 ethanol: acetic acid (
see
Note 6
).
2. Ethanol: 50, 70, and 100 % (v/v).
3. Ethanol: isopropanol: 50:50.
4. 100 % Isopropanol.
5. Melted paraffi n chips (
see
Note 7
).
2.3 OCT Embedding
1. Farmer's fi xative: 3:1 ethanol: acetic acid. (
see
Note 6
).
2. 10 % Sucrose solution: 10 % sucrose (w/v) in phosphate-
buffered saline (PBS;
see
Note 9
).
3. 15 % Sucrose solution: 15 % sucrose (w/v) in PBS (
see
Note 9
).
4. OCT (optimal cutting temperature compound) or CMC
(carboxymethylcellulose).
5. Membrane slide (
see
Note 8
) or plain slides.
6. 70 % Ethanol: store at −20 °C.
7. Ethanol: 95, 100 % (v/v).
8. 100 % Xylene.
3
Methods
3.1 Fixation and
Embedding of Plant
Tissues for Paraffi n
Sectioning
1. Most frequently acetone [
13
,
14
] or Farmer's fi xative (3:1
ethanol:acetic acid) [
8
,
11
,
15
,
16
] are used for fi xation as they
provide the best results for a large number of different tissues.
Plant tissue samples are typically fi xed at 4 °C between 1 and
24 h (
see
Note 10
).
Search WWH ::
Custom Search