Spinal cord dissections take a maximum of 15 min from decap-
itating the animal and brains can be retrieved in less than 3 min.
For storage of particularly brittle tissues, e.g., spinal cords from
neonatal mice, use small tubes. Other tissues, e.g., brains, can
be stored in aluminum foil.- Embed thin, brittle tissues, e.g., spinal cords, in cryo embed-
ding medium (OCT) to avoid tissues from rupturing during
sectioning. Avoid creating bubbles in the OCT (as these will
render the sectioning uneven). - The thickness of the tissue sections depends on the size and
shape of the cells of interest. To ensure that one cell is spanning
the entire depth of the section, use a section thickness equal to
the minimalradiusof your cells as a guideline. Only capture
cells that have a visible nucleus surrounded by cytoplasm. This
will maximize the RNA content of your sample and minimize
the risk of collecting several cell layers hidden underneath your
cell. - When placing tissue sections on membrane LCM slides, avoid
touching the membrane with sharp objects like forceps or cryo-
blades. The membrane is highly fragile and keeping it intact is
crucial for good staining and drying of the slide for LCM. - Sectioned tissue can be stored for several weeks on slides prior
to performing LCM. However, faster processing is recom-
mended for better staining and LMD cutting outcomes. - When staining sections from OCT embedded tissue, carefully
agitate the staining containers at every step to ensure that the
OCT is properly removed. - The incubation time with HistoGene and cresyl violet staining
solution is variable for different tissues and cell types. Deter-
mine the optimal staining time by starting with 20 s incuba-
tion. Prolong the incubation time if necessary, but keep it as
short as possible to minimize RNA degradation. Additionally,
in our hands cresyl violet seems to better visualize the nuclei of
small glial cells that surround the neurons, enabling separate
isolation also of these. - Acetone should be fresh for every staining and ice cold
before use. - The primary antibody for the rapid staining is used at a high
concentration. For example, we use the sheep-anti TH anti-
body at a 1/25 dilution for LCM while we normally utilize it at
1/1000 for immunohistochemistry and confocal imaging. In
addition, the antibody needs to show a high degree of specific-
ity to recognize only the protein and cells of interest. To
increase the versatility of LCM-seq, we have tested and shown
that an increased incubation time for the primary antibody for
up to 1 h still results in sequencing data of high quality [1].
108 Susanne Nichterwitz et al.