Figure 31 Part of collected samples from leaf 1, green stem 2, lignified stem 3, and
taproot 4
Table 3 Composition of stock solutions
Final solution Composition
Quantity of stock solution
Solution A Anhydrous NaH
2
PO
4
MW 120 2.4 g
Distilled H
2
Up to 100 mL Solution B
Anhydrous NaH
2
PO
4
MW 142 2.84 g
Distilled H
2
Up to 100 mL Buffer phosphate pH 7.2; 0.2 M
Solution A 28 mL
Solution B 72 mL
Fixative solution Buffer phosphate pH 7.2; 0.2 M
50 mL Paraformaldehyde 20
10 mL Glutaraldehyde 50
2 mL Caffein
1 g Distilled H
2
Up to 38 mL
2.9.2 Softening procedure
For big and hard samples, a pre-treatment was necessary. First, samples were put in histology cassettes. The name of samples was written on the cassette by pencil. Cassettes
were rinsed by H
2
O for a while and after were put in softener solution DEAM solution, the composition can be seen on Table 4 in specific Erlenmeyer with stirrer. After, the samples
were put in HISTOS 5 instrument Mileston Srl, Sorisole, Italy with special program, softening, for one hour, at 80
o
C PHIV platform, CIRAD, Montpellier, France.
Table 4 Composition of DEAM solution
Solution Quantity
H
2
O 89 mL
Glycerol 10
Tween 20 1
.
Figure 32 Preparation of samples before and after using HISTOS 5. A Samples kept in
fixative solution, B samples in histology cassette, C samples in specific Erlenmeyer for HISTOS 5 with stirrer, D the rapid microwave histoprocessor
HISTOS 5, E samples with Erlenmeyer put in HISTOS 5, F the monitor of HISTOS 5 for controlling the process, G samples in vacuum, H, I embedding
process with resin solution, J preparation a plastic mould with resin solution, K the samples in the well, K the samples covered by resin solution with the
desirable orientation
2.9.3 Tissue processing dehydration and impregnation
All steps of dehydration, dehydrationimpregnation, and impregnation were carried out in the rapid microwave histoprocessor HISTOS 5. Each step can be seen on the Table 5.
This procedure was modified for rubber.
Table 5 Stages of dehydration, dehydrationimpregnation, and impregnation of samples by HISTOS 5
Step Treatment
Duration of step
min Number of cycle
Standard for PHIV
Modified for rubber
1. Dehydration
Ethanol 70 8
1 1
Ethanol 95 8
1 1
Ethanol 100 8
1 1
2. DehydrationImpregnation
Ethanol 100 8
1 1
EthanolButanol 1:1 8
1 1
Butanol 100 13
1 1
3. Vacuum
30 1
4. Impregnation
ButanolResin 18
1 2
Resin 90
1 2
Estimation of total times is around 5 hours.
2.9.4 Embedding process
