- The secondary antibody should be of high quality and used at a
high concentration to minimize incubation times and thus
unnecessary degradation of tissues. Here, we use a 1/25 dilu-
tion of an antibody that is used at 1/500 in standard immu-
nohistochemistry applications. - The relative humidity during LCM for downstream RNA pro-
cessing should optimally be between 20 and 40%. RNA degra-
dation increases with rising humidity whereas low humidity can
increase statics and hamper the collection process if using a
collection system based on gravity. However, we have success-
fully processed samples collected at a relative humidity between
10 and 60% and at temperatures between 20 and 29C. We
recommend taking notes of temperature and relative humidity
to enable a correlation of these parameters with sample quality.
Furthermore, the use of anti-statics devices such as the Milty
Zerostat gun may improve collection of cells. Note that also
the choice of PCR tubes may have an influence on statics and in
our hands, PCR soft tubes from Biozym Scientific work well. - We highly recommend saving microscope images of your
stained sections pre-LCM at 10 or 20magnification as well
as post-microdissection as a reference. Pre-microdissection
images can be helpful to identify cells with a clear nucleus and
nucleolus, as the contrast at 40magnification can be low. - Optimal laser settings depend on many factors including dry-
ness of the slide, humidity in the chamber, thickness of the
tissue, and tissue morphology, but also on personal preferences
and may be adjusted throughout the LCM process. As a guide-
line, adjust the laser power and aperture from low to high at the
beginning of every session. Move to the “no cap” position and
start outlining an area outside of your region of interest, but
with similar tissue morphology. Adjust laser settings until the
cutting line is neat and thin and the membrane with the tissue
detaches from the slide. Decreasing the speed of the laser will
aid in keeping the cutting line thin as lower laser power and
aperture are needed. When using a system based on gravity
(such as Leica’s), inspect the collector after isolating an easily
countable number of cells to get an estimate of the actual
number of cells that are collected into the cap. Keep in mind
that statics can vary significantly throughout a session. Check
the collector regularly if collecting large numbers of cells over
several hours. - The volume of lysis buffer depends on the number of cells
collected; for 50–150 cells, we use 5μL. Do not pipette the
lysis buffer up and down when collecting a very small number
of cells (up to 10), to avoid losing cells in the pipette tip.
Furthermore, we recommend to place the tube back into the
Spatial Transcriptomic Profiling Using LCM-seq 109