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#  ARABIDOPSIS  #   Procedure was performed in UCDavis
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>> Harvesting Seeds

Arabidopsis Plants were grown in a Conviron chamber under continuous
light with fluorescent lamps at 20°C and 50% - 70% relative humidity.
Along the shoot, a linear correlation was observed between the developmental
stages and the length of Arabidopsis siliques. Independent silique
replicates at appropriate developmental stages were collected during the
afternoon and the seeds were staged by viewing whole mount seeds using Normarski microscopy. 

>> Seed Fixation and Embedding

Seeds were either dissected out of siliques or left in sub-divided
siliques and fixed in 3:1 (v/v) ethanol to acetic acid. Fixation was
performed for 4 hours at 4C, washed 3 times with 70% ethanol for 5
minutes each and kept in 70% ethanol overnight. Seeds were dehydrated
with a series of ethanol solutions with increasing concentration (85%,
95%, 100%) for a minimum of 1 hours each. Seeds were then infiltrated
with different xylene dilutions (in ethanol)(25%, 50%, 75%, 100%) for a
minimum of 2 hours each. Finally, seeds were incubated approximately 3-4
times with melted paraffin at 60C for a minimum of 3 hours and embedded
with paraffin in metal boats and kept at 4C until further use. Each
block contains 30-50 seeds.

>> Seed Sectioning

Arabidopsis seed paraffin sections of 5 um and 7 um were generated.The
slides were kept in a oven at 42C overnight.

>> LCM

The PEN-Foil slides containing the paraffin sections were treated with
xylene solution in order to melt the paraffin. Two incubations of two
minutes each were performed in the fume hood, where the slides were kept
at room temperature until the following day. The slides were kept in a
slide holder at room temperature to minimize exposure to humidity.

We used Leica LMD6000 system to capture the different seed regions. For
each slide, the sections meeting the criterion for each compartment were
selected, a picture of the region to be captured was taken, the region
was captured by LCM, and a final picture was taken documenting the
captured area. Captured material was collected on a 0.5 ml eppendorf
tube cap containing 30 ul of RNAqueous lysis buffer from the Ambion
RNAqueous-Micro kit. After collecting all samples from a particular seed
region on a slide, the tube containing the captured material was
immediately spun down and transferred to dry ice. At the end of the
session, tubes containing the collected material were placed at -80°C
until further use. As an example, we collected 895 sections for one
replicate of Peripheral Endosperm, 376 sections for one replicate of
General Seed Coat.