Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 14

21. To clean the knife/blade, use petrol; this is not as dry and nox-
ious as xylene or toluene. Do not touch the fi ne edge of the
knife/blade as it can be very easily damaged.
22. Cut sections into a ribbon (Fig. 2d ). As it is always more diffi -
cult to cut the fi rst section, it is easier to cut ribbon than indi-
vidual sections. It is also easier to arrange pieces of ribbon on
slides. Speed of the cutting stroke should be adjusted accord-
ing to paraffi n and temperature. Slow and steady strokes usu-
ally result in best sections with least compression. Use
moistened brush to manipulate ribbons, as it is easier then for-
ceps and less probable to cause blade damage. For trouble-
shooting of the most common problems ( see Table 3 ).
23. Transfer ribbon on black cardboard and cut it into equal pieces
to be placed on the glass slides. Their length should be less
then length of available cover slips. If series of sections is
required, take care to maintain their proper order. Nick in the
paraffi n block, which can be seen in the ribbon, can make
proper orientation easier. There are two sides of the ribbon.
The glossy one should be placed towards the slide (down),
while the matt site is the upper one.
24. Use pre-coated slides to ensure adhesion of sections for further
manipulation ( see Note 4 ). Cover the glass slide with distilled
water so that only small part stays without water and can be
used to handle the slide. The surface tension of water helps to
fl atten sections, and enough of free space surrounding ribbons
should be available.
25. Float the ribbons on the water surface, arrange it, and heat it
on hot plate to stretch and fl atten the sections (Fig. 2e ). The
temperature of the plate should be approx. 5 °C lower than
paraffi n melting temperature. Let the slides on the plate for
5-10 min, as stretching the ribbon should be slow and gradual
to be effi cient. Temperature can be adjusted also experimen-
tally so that it is gradually increased till the paraffi n of sections
melts, then the temperature is adequately lowered. If the tem-
perature is too high, the ribbons will melt (objects are lost); if
too low, fl attening does not get complete (lines and wrinkles
are still discernible on ribbons). Stretching of sections in water
bath is more convenient for large individual sections. If small
bubbles form under the ribbon, use boiled distilled water to
eliminate dissolved gasses.
26. Remove the slide from hot plate, let it cool down, and rear-
range the ribbons if necessary.
27. Gently remove most of the water and let the slides dry to attach
sections to the slides on warm plate (40 °C overnight). Protect
slides from dust.
28. When dry, sections can proceed to staining or store the slides
in box before further processing.