Is
it necessary to dehydrate the sample on ethanol and xylene?
The final dehydration and xylene steps are absolutely crucial
for successful LCM. In some cases where excess water is a
problem, it may be necessary to extend the dehydration and
desiccation times to 10-30 minutes prior to using the material
for LCM. Any moisture present in the sample during LCM will
impede transfer efficacy. A benefit of complete sample dehydration
is the protection of RNA quality. The HistoGeneTM LCM Frozen
Section Staining Kit, available from Arcturus, optimizes the
tissue preparation process to preserve the integrity of nucleic
acids.
Why
won't my cells come off the slides and what can I do about
it?
Perhaps the single most important procedure towards ensuring
good LCM is dehydration of the sample upon completion of the
sample preparation and processing. There is a difference between
dehydrating and drying. Dehydration is an active removal of
water molecules while drying is a passive process that brings
the tissue to equilibrium with the environment. The presence
of water within the tissue sample will not allow proper wetting
of the transfer film or good capture of the cell.
Proper dehydration entails the use of graded ethanols. A final
100% ethanol step is of great importance, but it will only
be effective if 100% ethanol that has been freshly dispensed
is used. Due to the hydroscopic nature of ethanol, moisture
from the air can hydrate the 100% ethanol, making it less
effective as a dehydrating agent. Xylene incubations will
completely remove ethanol and water remnants from the sample.
Air drying of the slides will remove the xylene from the sample;
however, the xylene must be completely evaporated. Although
a sample may appear completely dehydrated after a short drying
period, xylene may still remain. To ensure complete dehydration,
a slide should typically be air dried for a minimum of 5 minutes
under a fume hood. If the humidity of the laboratory is an
issue, such as during summer months of tropical environments,
the drying ca take place within a vacuum desiccator. Also
longer exposure to fresh 100% ethanol and xylene have shown
to aide in the transfer efficiency.
The final dehydration and xylene steps are absolutely crucial
for successful LCM. Any moisture present in the sample during
LCM will yield less than optimal results. Hence, no dehydration=no
LCM. Microdissection may be performed without staining, but
dehydration is mandatory.
Can
I store slides that I have processed and dehydrated for future
use?
Yes, but it is only recommended if you plan to extract DNA.
The slides should be stored in a desiccator at room temperature.
Keeping the sample dehydrated is crucial to ensure a successful
microdissection for future use. This is not recommended if
you plan on doing any RNA or protein experiments. Storage
of already stained/dehydrated specimen slides may severely
compromise RNA/protein quality/quantity.
Is
it okay to use RNA preservatives to preserve my samples?
There have been reports that the use of RNA preservatives
as a pre-treatment fixative before embedding, presents problems
with the integrity of the frozen tissue block. The ethanol
in the RNA preservatives inhibits the tissue biopsy from freezing
thoroughly within the frozen-embedding medium, thereby affecting
section morphology. Thus, the use of RNA preservatives is
not recommended for preparation of LCM samples.
I
stored a sample after successfully performing LCM. When I
used the sample later, I was unable to perform LCM. What do
I do?
The sample most likely picked up some moisture between the
first and second LCM sessions. The sample can be dehydrated
again through an ethanol and xylene series. Perform two 30-second
dips in 95% ethanol, followed by one 30-second dip in freshly
dispensed 100% ethanol, followed by one 5-minute xylene dip.
Allow the sample to dry a minimum of 10 minutes in a fume
hood or vacuum desiccator before proceeding back to LCM. These
reagents are provided along with the HistoGene kit. However,
RNA and protein quality may be compromised.
How
do I transport my sample from one location to another?
Sample slides that have been processed and dehydrated should
be stored in a slide box with a pouch of desiccant to keep
the sample dry. If frozen sample is to be taken out of a -80c
freezer, then it must be transported in an adequate amount
of dry ice.
I
like to dry my frozen tissue section onto the slide to ensure
that the section is adequately mounted on the slide. Can I
still get good LCM?
Drying the section onto the slide reduces the ability to get
optimal LCM. The sample should either be frozen at -80 or
fixed immediately after removal from the cryostat. If you
wish to have greater adhesion of your sample to the slide
due to concerns that it will come off during staining protocols,
consider the use of charged, poly-L-lysine-coated, or silane-coated
slides.
Is
it necessary to stain your slide for LCM?
No. However staining improves tissue visualization and several
stains have been used successfully.
What
stains do you recommend?
Arcturus recommends the HistoGene(TM) LCM Frozen Section Staining
Kit, which comes complete with all the reagents and supplies
needed for preparing frozen tissue sections for LCM. Dehydration
and staining solutions, slide jars - even specially coated
glass slides - are included, along with a detailed, step-by-step
protocol. LCM certified, the HistoGene Kit ensures consistent
preparation of quality samples ready for laser capture.
In
addition LCM has been known to work with a wide variety of
pathological stains.(i.e. Hematoxylin and Eosin, Methyl Green
and Toluidine Blue). However, staining tissue for a long period
of time may reduce the efficiency of transfer and quality
of nucleic acids obtained. Hematoxylin may bind to nucleic
acids and adversely affect downstream research processes.
Hence, Arcturus recommends Histogene as a general-purpose
rapid frozen tissue stain staining for LCM.
To
ensure high quality of RNA/protein recovery/integretity, fresh
clean solutions are highly recommended to be used each time
when preparing tissue sections for LCM.
Is
it possible to use ImmunoHistoChemically stained slides (IHC)?
Yes. The combination of IHC-labeled slides and LCM provides
scientists with powerful new capabilities. Please refer to
Immunohistochemical
Staining (IHC).
What
range of tissue thickness do you recommend?
Arcturus recommends 7-8 microns as a standard range of thickness
for tissue sections.
Any
special tips when processing Frozen Tissue sections versus
Paraffin Embedded Tissue (PET)?
The Arcturus LCM systems work well with both types of tissue
preparation. Frozen sections require attention to the speed
in which you take the section from the water/staining steps
into ethanol. There should be no delay in any of these steps.
With both types of tissue, it is important to pay attention
to dehydration and desiccation of the material prior to LCM.
Some users increase the time of the final xylene wash to 10
minutes.
Any
special tips when working with Formalin-fixed versus Ethanol-fixed
tissue?
We have seen excellent LCM from Formalin-fixed tissue, although
the DNA yield decreases with prolonged fixation times, and
quality of RNA is severely compromised. When using Formalin-fixed
tissue, 2.5 - 4 hours for fixation is recommended.
How
do I get best recovery of RNA?
RNA recovery is best performed on freshly frozen tissue. All
solutions must be RNAse free and the tissue must be handled
quickly to prevent any endogenous nucleases in the tissue
from degrading the RNA. PicoPure RNA Isolation kit available
from Arcturus provides an optimized process for efficient
recovery of total cellular RNA from small amounts of sample
such as LCM.
What
parameters do you suggest regarding protein recovery following
LCM?
For optimal protein recovery, freshly frozen tissue is highly
recommended. Best extraction appears to be with a buffer that
contains detergents and chaotropic agents.
Is
there any damage to the genetic material from the LCM process?
No. Data indicate that the LCM process does not impact the
recovery of the intended DNA and RNA from microdissected tissue.
The thermal transients seen by the tissue are mild and last
only a few milliseconds. The DNA, RNA or protein content of
the cell is not altered in any measurable fashion. There is
no published evidence of IR damage to nucleic acids unlike
the scores of papers reporting UV damage.
What
is the minimum number of cells necessary for molecular analysis?
Although the number of cells is dependent on tissue thickness
and type, results indicate that for DNA amplification, 10-20
cells from a 8µm thick section (in a single PCR* reaction)
are sufficient. For protein analysis, using 50,000 cells for
2-D gel analysis has been a successful starting point. For
Western Blot analysis, the number of cells required is approximately
2,000 to 3,000 cells. For RT-PCR from as few as 10 cells and
microarray analysis can be performed on 250 cells, provided
that the RiboAmp RNA Amplification kit is used.
What
slides should be used for optimum LCM?
Arcturus recommends the use of Histogene slides, which are
specially coated glass slides that offer a balance between
transfer efficiency and tissue adhesion.
Can
LCM be done on plant tissue?
Yes. High-quality gene expression analysis utilizing LCM has
been performed successfully on plant cellular material. Click
here for citations of published studies.
Do
you recommend using freshly prepared ethanols for dehydration
before LCM?
Yes. Any water in the final 100% ethanol bath will prevent
complete dehydration and result in poor transfer.
What
microcentrifuge tubes fit LCM caps?
SealRite® Thick-Walled Tubes (#1605-0000) from USA Scientific, Inc. have been demonstrated to fit the CapSure and the ExtracSure Sample Extraction devices and recommended for use with the Arcturus extraction protocols. If PCR is to be performed directly on the sample lysate, it is recommended to use GeneAmp™ 500 µl Thin-walled PCR* Reaction Tubes (# N801-0611) from Applied Biosystems, which also fit the CapSure and the ExtracSure Sample Extraction devices.
*
Polymerase Chain Reaction (PCR) is a patented process.
Arcturus does not encourage or support the unauthorized or
unlicensed use of the PCR process. Use of this product
is recommended for persons that either have a license to perform
PCR or are not required to obtain a license.
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