to the minimal value sufficient to cut through the membrane,
and we suggest having at least two laser cut repetitions (i.e., the
laser will cut the same area twice). This is critical to ensure that
the membrane is properly cut from the rest of the samples since
these are the samples of interest that will need to stay attached
to the chamber after removal of the ring (in our case, 75–85%
of laser power was enough).
7. Using the proper objective (depending on the size of the
cellular protrusions of interest) [27], you can select the ROIs
by manually drawing a line with a digital pen, directly on the
computer screen, around the protrusions of interest (Fig. 3
shows different types of cellular protrusions that can be isolated
by LCM).
8. We recommend selecting 10–100 ROIs at a time, prior to
cutting, but these can be changed according to need or user
preferences.
9. Between cutting sessions, replace the PBS with fresh PBS to
remove any dead cells or debris and store the dish at 4
C until
further use.
10. The dish can be used for 4–5 days approximately. Thus, as
many ROIs should be isolated as possible per day and a new
dish needs to be prepared if more ROIs are required after
5 days post-fixation (see Note 13).
ä
Fig. 3 (continued) imaged at Â20 (a), Â40 (b–d), or Â60 (e) magnifications.
Various types of cellular protrusions are shown. For all cases, (i) ROIs are drawn
around the protrusions of interest, (ii) are representative images of cellular
protrusions after the laser cut, and (iii) are images of the desired isolated cellular
protrusions after removal of the LCM membrane containing the “unwanted”
cells. Examples of different types of protrusions are shown: (a) axons and
dendrites from dCADs are shown (scale bar ¼ 100 μm). Small dendritic filopodia
can be observed (black arrows); (b) in order to increase the number of cellular
protrusions, CAD cells were treated for 5 min with 100 μM H 2 O 2 prior to fixation.
Various types of cellular protrusions are shown (scale bar ¼ 10 μm). Individual
subtypes of cellular protrusions can be specifically isolated such as (c) GCs
(scale bar ¼ 20 μm), (d) filopodia (scale bar ¼ 10 μm), and (e) TNTs (scale bar
top ¼ 50 μm; bottom ¼ 10 μm). TNTs do not touch the substratum, and tension
is visible within these structures (ei). As expected, after being cut, the structures
collapsed onto the LCM membrane (eii,iii), clearly demonstrating that these
protrusions were TNTs, and not attached filopodia or other types of protrusions.
(Reproduced from ref. 27 with permission from International Journal of Molecular Sciences)
36
Ana Gordon and Karine Gousset
and we suggest having at least two laser cut repetitions (i.e., the
laser will cut the same area twice). This is critical to ensure that
the membrane is properly cut from the rest of the samples since
these are the samples of interest that will need to stay attached
to the chamber after removal of the ring (in our case, 75–85%
of laser power was enough).
7. Using the proper objective (depending on the size of the
cellular protrusions of interest) [27], you can select the ROIs
by manually drawing a line with a digital pen, directly on the
computer screen, around the protrusions of interest (Fig. 3
shows different types of cellular protrusions that can be isolated
by LCM).
8. We recommend selecting 10–100 ROIs at a time, prior to
cutting, but these can be changed according to need or user
preferences.
9. Between cutting sessions, replace the PBS with fresh PBS to
remove any dead cells or debris and store the dish at 4
C until
further use.
10. The dish can be used for 4–5 days approximately. Thus, as
many ROIs should be isolated as possible per day and a new
dish needs to be prepared if more ROIs are required after
5 days post-fixation (see Note 13).
ä
Fig. 3 (continued) imaged at Â20 (a), Â40 (b–d), or Â60 (e) magnifications.
Various types of cellular protrusions are shown. For all cases, (i) ROIs are drawn
around the protrusions of interest, (ii) are representative images of cellular
protrusions after the laser cut, and (iii) are images of the desired isolated cellular
protrusions after removal of the LCM membrane containing the “unwanted”
cells. Examples of different types of protrusions are shown: (a) axons and
dendrites from dCADs are shown (scale bar ¼ 100 μm). Small dendritic filopodia
can be observed (black arrows); (b) in order to increase the number of cellular
protrusions, CAD cells were treated for 5 min with 100 μM H 2 O 2 prior to fixation.
Various types of cellular protrusions are shown (scale bar ¼ 10 μm). Individual
subtypes of cellular protrusions can be specifically isolated such as (c) GCs
(scale bar ¼ 20 μm), (d) filopodia (scale bar ¼ 10 μm), and (e) TNTs (scale bar
top ¼ 50 μm; bottom ¼ 10 μm). TNTs do not touch the substratum, and tension
is visible within these structures (ei). As expected, after being cut, the structures
collapsed onto the LCM membrane (eii,iii), clearly demonstrating that these
protrusions were TNTs, and not attached filopodia or other types of protrusions.
(Reproduced from ref. 27 with permission from International Journal of Molecular Sciences)
36
Ana Gordon and Karine Gousset