Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 8

pour the solvent into a fl ask and introduce enough of desiccant
(approx. 1/5 of volume). Desiccant can be either solvent-
drying molecular sieve (3 Å for both butanol and ethanol) or
anhydrous salt (e.g., K 2 CO 3 , CaSO 4 , or CuSO 4 ), which binds
the water but does not dissolve in alcohol. Let the capped fl ask
stand overnight. Filtrate or decant water-free solvent and keep
it in tightly closed fl ask to prevent air humidity entrance. To
regenerate the molecular sieves as well as hydrated salt, place
them into drying oven at 250 °C in thin layer for about 2 h.

3. Paraffi n: paraffi n is a mixture of long-chain alkanes. There are
various types of paraffi n suitable for embedding and sectioning.
The classical method is based on recycling of suitable paraffi n
and alchemy of preparation of such paraffi n [ 29 ]. Most labora-
tories use commercially available and easily accessible paraffi n
these days. Various brands are on the market (e.g., Paraffi n,
Tissue-Tek, Paramat, Paraplast, Histoplast, Sasol; see Note 3 ).

4. Gelatine-subbing coated microscopic slides (alum gelatin
adhesive, chrome alum; [ 41 ]): Place 0.5 g of pure gelatin in
100 ml of distilled water and heat to approx. 45 °C to dissolve
it completely. Add 0.05 g of KCr(SO 4 ) 2 .12H 2 O (the usage of
other alums is also possible) and dissolve and fi lter the solu-
tion. Immerse set of clean slides in staining rack into the solu-
tion for 10 s, blot excess of solution, and let the slides dry
(48 h at room temperature or 12 h at 50 °C); protect slides
from dust. Slides can be submerged several times (2-5 times)
to heighten coating layer if necessary ( see Note 4 ).
5. Poly-l-lysine-coated slides: Dilute 10× Poly-l-lysine stock solu-
tion (0.1 % w/v) to prepare working solution. Immerse clean
slides into the solution for 10 min to 1 h. Dry and store coated
slides in dust-free dry place; 4 °C is recommended for longer
storage ( see Note 4 ).
6. Glycerol albumen: Mix carefully egg white with equal volume
of pure glycerol. Filter the mixture over glass wool or few lay-
ers of gauze. Add 1 % of sodium salicylate or thymol as a pre-
servative (causes background autofl uorescence!). Alternatively
use 0.5-1 g NaN 3 (be careful, toxic). Smear a tiny amount
(pinhead volume) evenly over a clean grease-free slide with
your fi nger to make very fi ne (not wet) coating. Protein pre-
cipitate forms on the slides if high amount of albumen adhesive
was used ( see Note 4 ).

1. High-viscosity cryoembedding medium ([ 39 ]; see Note 5 ):
Dissolve 65-75 g of polyvinyl alcohol (PVA) 56-98 in 1 l of
distilled water or phosphate buffer (pH 7.4; 50 mM). Warm
up to 100 °C to completely dissolve it. Add 10 ml of Tween
20, 0.5-1 g NaN 3 (preservative for long-term storage) and
40 ml polyethylene glycol 400. Optionally supplement

2.4 Cryosections