Biology Reference
In-Depth Information
non-reproducible inconsistencies in the GUS patterning could
sometimes be observed during repetitions.
15. The 80 % ethanol will stop the GUS staining, fi x the plant
material, and begin its bleaching by chlorophyll extraction.
16. The solution containing HCl digests cell walls and ensures
elasticity and transparency of the plant tissues. The samples
will start to become sticky and might adhere to the pipette tip
during removal of the HCl solution. Be careful, therefore, to
avoid damaging the specimen; use tweezers whenever neces-
sary. Work carefully and use gloves when handling clearing
solutions.
17. NaOH solution is used to stop the HCl-mediated degradation
process by neutralization. Work carefully and use gloves when
handling clearing solutions.
18. The protocol can be interrupted here and samples stored in
50 % glycerol for up to 6 months in parafi lm-sealed staining
plates. Check the amount of 50 % glycerol during storage to
avoid samples' drying during storage.
19. According to the expected segregation ratios, recessive homo-
zygous mutation appears at frequency of approximately 1:7
(assuming only one of the two initials in the SAM is mutated
and the recessive mutation segregates according to Mendelian
rules).
20. Use a small volume of mounting media, as this can increase
the quality of the resulting virtual slide because all the seed-
lings will be in the same plane as defi ned by the thin layer of
mounting media. We routinely use 100
l of 50 % glycerol for
22 mm × 50 mm cover slips. Using a standardized volume of
mounting media ensures the comparable quality of all samples.
Avoid excessive spattering of the slide label area, as this com-
monly is responsible for any inaccurate handling (loading
problems) by the automated microscope and for damage to
specimens and/or the automated microscopy system. When
following the instructions stated in our protocol, fi xing the
cover slip using tape or nail polish is not necessary. Avoid the
presence of bubbles during the specimen preparation. Use
mounting media (50 % glycerol) prepared separately, as using
50 % glycerol from the staining plate will ultimately increase
the amount of contaminants and bubbles in the specimen and
can signifi cantly decrease the quality of the fi nal image.
21. Samples can be stored for a long time in a refrigerator (at
4 °C). According to our experience, storing the samples for up
to 6 months in a horizontal position and in a plastic slide
holder does not negatively affect the specimen quality. Avoid
storing the samples in paper slide holders or in a non-horizontal
position. Both lead to leakage of glycerol and specimen
μ
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