- Master mix 5 (Index ligation and enrichment PCR): 10μL5
high fidelity PCR buffer, 1.5μL dNTPs, 1μL high fidelity PCR
enzyme, and 2.5μL ddH 2 0; 15μL per sample.
3 Methods
3.1 Tissue
Dissection
- Clean work benches and tools and prepare all materials needed.
- Anesthetize animals (using 5% avertin or other anaesthetic) and
decapitate. - Dissect tissues as rapidly as possible, place the tissue into a small
plastic spoon (cut from PE pasteur pipette) and snap-freeze by
submerging the tissue in 2-methylbutane on dry ice (seeNote 1). - Place tissues in RNase/DNase-free tubes or in clean aluminum
foil (seeNote 1) on dry ice for transportation and store at
80 C until further processed.
3.2 Tissue
Sectioning
- Clean work benches and tools with 70% ethanol and
RNaseZap. - If embedding of the tissue is required (seeNote 2) use pre-
chilled cryo embedding medium to avoid tissue thawing during
embedding. Place a labeled mold filled with OCT on an even
block of dry ice. Wait a few seconds until the OCT in the
bottom of the mold is frozen (turns white), but the top layer
is still liquid. Place the spinal tissues onto the bottom of the
mold using cold forceps. Leave the tissue block on dry ice for
5–10 min until it is completely frozen. Place the mold in the
cryostat to equilibrate to the sectioning temperature (wait at
least 30 min before sectioning). Remove the plastic mold and
trim away any excess OCT from the tissue block (using a razor
blade). Brains (and other larger tissues) do not need embed-
ding prior to sectioning. Mount the tissue or the tissue block
(embedded tissue) on a chuck with OCT and wait until it is
properly frozen. - Label LCM slides and place them into the cryostat in a plastic
slide box (to cool down) that is kept in the cryostat at 20 C
during sectioning. - Section tissues at 20 C at the required thickness (seeNote 3).
Place tissue sections on membrane glass LCM slides. To better
attach sections, “melt” the tissue onto the LCM slide by plac-
ing a finger or a metal heat stick on the back of the slide just
underneath the section (seeNote 4). - Store slides with tissue sections at 80 C until further pro-
cessed (seeNote 5).
100 Susanne Nichterwitz et al.