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
After all samples were processed by HISTOS 5 the next step was embedding with resin solution Technovit® 7100, Heraeus Kulzer GmbH, Wehrheim, Germany. Resin
solution was made by adding 1 g hardener I 1 bag to 100 ml Technovit 7100. For embedding, 1 mL Harderner II was added to 15 mL prepared resin in a Falcon tube and after
the solution was gently mixed for homogenous. A plastic HistoMold Leica, Nussloch, Germany was prepared before. The sample name was noted on the plastic mould by pencil.
First, a little volume of resin solution was poured in the wells, and then each sample was gently and slowly put in the well with the desirable orientation. Resin solution was added
gradually until all samples covered by resin before hardening because of the polymerization process. Samples embedded in resin were put in the oven 37
o
C for polymerization achievement and long storage. All work was conducted under the fume hood PHIV platform,
CIRAD, Montpellier, France.
2.9.5 Sample specimen preparation
Resin-embedded specimen was ready to cut by automated rotary microtome Leica RM2255 Leica, Nussloch, Germany. Before resin-embedded specimen was excluded from
the mould, the surface of preparation was scratched by pinsetter or scalpel. The rough surface was covered by very strong glue, affixed on the support, and ready for cutting. The cross-
sections obtained from microtome were thin slices 3.5 m. Each thin slice was put on the
surface of water in a big dish. Slices of specimen were put on glass slides immersed in water then set on a hot plate 40
o
C for 30 min. Slides were dried and mounting solution glycerol: water = 50:50 was used to fix coverslips. The edge of coverslips was covered by polish
solution or Pertex xylene 40-60 and ethylbenzene 10-20 Gothenburg, Sweden for automatic microscopic scanning. The glass slides must be soaked first in alcohol 100 before
used them for removing fat traces. 2.9.6 Sample staining
There were two staining used, first was Oil Red O-Toluidine Blue staining method adapted from Lillie and Ashburn 1943 by PHIV platform CIRAD, Montpellier, France.
The composition of staining was Oil Red O 0.5 in alcohol 70 and Toluidine-Blue 0.1 in Walpole buffer 0.1 M pH 4.2. The solution of Oil Red O was filtered before use by the
membrane filter. Slides of samples were stained by Oil Red O for 10 minutes and rinsed by H
2
O. Furthermore, slides of samples were stained by Toluidine Blue for two minutes and then rinsed by H
2
O. The other staining was periodic acid-Schiff and Naphtol Blue Black NBB, Fisher
1968 adapted by PHIV platform CIRAD, Montpellier, France. The different components of staining were periodic acid solution 1 in water distilled prepared immediately upon before
staining, Schiff solution, and NBB NBB 1 g, acid acetic 7 mL and H
2
O was added until 100 mL Sigma-Aldrich, St. Louis, USA. Slides with sections were hydrolysed freshly with
periodic acid at room temperature for 5 minutes. Slides were washed by tap water and distilled water then dried on tissue paper. Slides were stained by Schiff’s reagent in darkness
in fume hood at room temperature for 10 minutes and washed by running water until the water was colourless and continued with distilled water quickly. Microscopic control was
done after washing with water. Before used it, NBB solution was filtered. Slides were stained in NBB solution at 50
o
C for 5 minutes. After few rinses coverslips were sticked to slides using Isomount 2000 Labonord, Templemars, France as mounting medium.
2.9.7 Slide observation, qualitative, and quantitative parameters
The slides were analysed by digital slide scanner NanoZoomer 2.0-HT Hamamatsu, Japan at The Institute for Neurosciences of Montpellier INM. The image bar scale was
defined using image analysis software ImageJ, Bethesda, Maryland, USA. Parameters of qualitative and quantitative for leaf, green stem, lignified stem, and root can be seen in Table
6. Table 6 Qualitative and quantitative parameters for slide observation
Organ Part
Parameter Qualitative
Quantitative
Leaf main nerve
starch St, polyphenol PO laticifer cell LC
lamina starch St, polyphenol PO
laticifer cell LC, palisade cell PC, stomata S, the width of cuticle C,
upper epidermis
UE, palisade
parenchyma PP, spongy parenchyma SP, and lower epidermis LE
Green stem bark, cambium,
xylem, pith starch St, polyphenol PO
the width of bark B, cambium Ca, xylem X, and pith P; primary laticifer
cells PLC, and laticifer ring LR
Lignified stem bark, cambium,
xylem, pith starch St, polyphenol PO
the width of bark B, cambium Ca, xylem X, and pith P; primary laticifer
cells PLC, and laticifer ring LR
Taproot R1 bark, cambium,
xylem, pith starch St, polyphenol PO
the width of bark B, cambium Ca, xylem X, and pith P; primary laticifer
cells PLC, and laticifer ring LR
2.9.8 Statistical analysis
