Working with the SL µCUT System from MMI AG
Protocols and more
2. Accessories
2.1. The membrane frame slide
The SL µCUT uses a metal frame slide with a 1.4µm PET-membrane. The advantage of this
membrane is the low auto fluorescence.
The handling of this slide is the same as a regular glass slide.
You mount the sample on top of the membrane, stain as usual, and then place the membrane
slide up side down on a glass slide; your sample will be in between the membrane and the
glass slide. This protects your sample from any contamination.
m embrane
m embrane
tissue
tissue
regular glass slide
tissue
alum inium fram e
alum inium fram e
alum inium fram e
m embrane
For better attachment you can coat the membrane with poly-L-Lysine or gelatine,
Placing the slide under UV light will also give the slide more adherence.
Do not use a cover slip!
2.2. The adhesive cap
The adhesive cap is a safe lock tube from Eppendorf with our special adhesive material in the
lid.
You place the cap on the membrane above the region of interest, and then you cut around the
cells or cell of interest. When the tube is lifted the cut out cells will adhere to the cap of the
tube.
To make sure that the cap is RNAse free you can place it under UV-light or incubate for some
hours in DEPC water at 60°C and dry it in a covered glass box in an oven.
3.What kind of Material can be microdissected with the SL µCUT?
With the SL µCUT you can microdissect sample from animals, humans or plants.
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paraffin material,
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cryo samples,
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cell smears,
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chromosomes
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living cells
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Cytospins
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Fine needle aspirates
You can extract DNA, RNA, Protein and chromosomes from histological samples and cell
cultures.
The system can also be used for selecting cloned live cells or microorganisms from culture.
4. Sample collection and preparation
4.1. Collecting the samples for RNA
4.1.1. Cryo samples
For RNA extraction, cryo samples are best used for good quality. The samples that are fresh
and quick frozen in liquid nitrogen are best for RNA extraction.
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Put a piece of tissue 0.5x0.5x1.0 cm in a cryo tube and put it in liquid nitrogen.
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After quick freezing you can transport sample on dry ice.
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Store it in a deep freezer at –80°C or in the vapour phase in a nitrogen tank.
4.1.2. Paraffin samples
Paraffin samples are not recommended for RNA extraction, but for DNA extraction it is fine.
You can modify the embedding procedure for RNA, but it is much better to use fresh frozen
tissue. For RNA you remove all formaldehyde-steps in the embedding procedure and use
shorter times in each bath. All the solutions have to be made from DEPC water.
You can use the regular embedding procedure; make sure that the pieces are not too thick.
Otherwise the fixation is not successful.
4.1.3. Cells or microorganisms
Put the cell or microorganism suspension in a centrifuge tube and spin this for 10 min.
(The correct revolution per minute for living organisms is described in the manual from the
centrifuge manufacturer.)
Wash the sediment two times in PBS-buffer solution pH 7.4 dissolved in RNAse-free water.
Decant the supernatant and add 2% Agarose (with one drop 0.1% Eosin Y for better contrast).
Mix the sediment and the agarose with a sterilized Pasteur pipette by intake.
Pipette the whole amount and make one large drop on top of Parafilm©.
After the drop has solidified, use it as if it were a normal tissue sample.
You can cut it as you would for a cryo sample or embedded in paraffin.
4.1.4. Cell smears
Before you can lay on smears you have to coat the membrane with Poly-L-Lysine, for better
attachment. Use the membrane slide as a normal glass slide, lay the smear and let it air dry or
fix it with 70% alcohol mixed with RNAse-free water.
The other way is to use a cytospin. This cytospin can also be used for micro organisms,
pleural infusion, urine, ascities or any other liquid.
For staining you can use your choice of stains. All Staining solutions have to make with
RNA-se free water.
4.2. Cutting cryo and paraffin samples, mounting and staining for RNA extraction
When you work with samples for RNA extraction, you have to work fast and in an RNAse
free environment.
The handling of samples for RNA extraction demands some modification from normal
histology lab preparation. All materials that you work with must be free of RNAses. Glass and
metals could be baked 3hours at 180°C. Plastics and sensitive materials could be cleaned by
UV-light, with sodium hyperchloride solution, or heating in DEPC water.
It is recommend that you work with a lab coat and gloves, and clean the workbench daily with
a product that gets rid of RNAse. Dust is very problematic, work in a closed environment
such as under a hood or in a Plexiglas box.
All staining solutions have to made up with RNAse free water.
Keep the steps that you need water for rinsing very short. Water is not good for working with
RNA. Modify the staining protocols to have short water steps, for example after staining in
hematoxylin wash the slide for a maximum of 15 seconds in water not for 5minutes.
Some times it is necessary to coat the membrane with poly-L-Lysine for a better attachment
of the sections.
4.2.1. Cryo samples
Cryo samples can be either OCT embedded or fresh frozen. If you embed your samples in
OCT you will have to use a 30 second water dip step to remove the OCT. If you choose not
to embed your samples you can use a drop of OCT to adhere your sample to the holder. Use
clean knifes and brushes or toothpicks to cut and move your sample. Samples should be cut
8µm thick, and care should be taken that no wrinkles or folds are present. When you place
the frozen tissue on the room temperature membrane, the sample should melt; you may want
to warm the section by placing a gloved finger on the membrane under the section, or using a
small brush to press the section onto the membrane.
After placing the sections on the membrane you can fix the tissue in 70% Ethanol for 30
seconds, other fixatives can also be used depending on the downstream applications.
4.2.2. Paraffin samples
Paraffin samples will be cut with a microtome. The thickness of the samples should be 5µm.
The sections should be floated in an RNAse free water bath. Put the slide in a slide holder
and let the sections dry for 3 hours at 58°C in a normal incubator or for1.5 hours when your
incubator has a blower inside.
After drying remove the paraffin with Xylene in 2 steps each step should be 5 minutes.
Rehydrate the slides in descending sequence of alcohol solutions. Stain your sample
according to your protocol.
4.3. Cutting cryo and paraffin samples, mounting and staining for DNA extraction
You can follow the instructions from the RNA extraction. DNA is more stable as RNA; there
is no need to use RNAse free reagents and instruments.
4.4 Plant cells
Cut the sections between 15-30µm, coat the membrane with Poly-L-Lysine. Put the paraffin
sections in the incubator overnight to dry. If the plant sections don’t adhere to the membrane
use a Celloidin treatment. After the 100% alcohol step from the rehydration dip the section in
2-3% Celloidin solution (Colloxylimun).
Wait for 2-3 min. and let it air dry. After drying do not use alcohol in the staining protocol,
except for the last steps. If working with unstained sections, dip only the slide into the
Celloidin solution.
Use the H&E staining with Eosin dissolved in water not in alcohol.
4.5. Living cells
When working with live cells, use our special cell chamber for microdissection. Let your cells
grown inside the chamber, the bottom is a poly-L-Lysine coated membrane.
To microdissect, place the cell chamber in the middle of our coated culture dish. This culture
dish has a thin adhesive coat on the bottom of it. This adhesive is coated with poly-L-Lysine.
Remove some of the medium and microdissect the cells of interest. Remove the cell chamber
from the culture dish and the cut out areas will be stuck on the adhesive material on the
bottom of the Petri dish. Now fill with your culture medium. After 1 or 2 days transfer the
cells to a regular culture dish or bottle for further growth. Sometimes it is useful to add some
gelatine; the gelatine will be used instead of feeder cells.
4.6. Immunohistochemistry in laser microdissection
Sometimes it is useful to use an immunohistochemistry method to find the exact region of
interest. For DNA extraction you can use your normal protocol, but for RNA extraction the
protocol has to be shortened, and the water steps must be RNAse free. The road to success is
using a one step method, this means after the incubation of your primary antibody you don’t
use biotin-avidin but use a labelled polymer antibody. Dako and Zymed offer this kind of
secondary antibodys. It is helpful to validate this method in your lab; this method may not
work for all applications. Add RNAse-inhibitor to the commercial antibodies. The right
concentration is described by the manufacturer. You can store your Immunofluorescence
stained slides in a clean slide box wrapped in aluminium foil to keep out light.
Cryo samples can be fixed with 70% Isopropanol including 1% beta-mercaptoethanol
dissolved in RNAse-free water at 4°C but not longer as 2 min. Do not use detergents such as
Tween 20 or Triton-X100. These detergents start the digestion process of the cell organelles
and damaged the epitopes.
If you immuno-stain slides that will be used from paraffin embedded material you need the
target retrieval. The SLµCUT system works with a metal frame membrane slide which can
not microwaved. Instead it is recommended to use a pressure cooker (5 min under pressure),
or an enzyme digest.
5. DNA AND RNA EXTRACTION FROM MICRODISSECTED MATERIAL
5.1. RNA Extraction
After micro dissection pipette 50µl GTC solution with 1% beta-Mercaptoethanol in your tube
and invert, vortex for 1 min and store it in this position on ice for 3 min.
Vortex once more and spin it slowly. The sample can be left overnight at this stage. If you are
using kit such as; Stratagene Absolutely RNA Nanoprepkit continue as described in the
enclosed protocol.
If you use the GTC buffer solution you can store this lysate for weeks in a –80°C deep
freezer.
Attention: You don’t lose RNA by doing the microdissection, you lose the RNA before and
after the microdissection step. The main point here being, proper handling of the sample in
your lab is very important. This means clean the instrument stage each morning with bleach
(25ml saturated sodium hyper chloride solution in 1 Litre water.). Wear a lab coat, change
your gloves frequently, and work in a “clean area. Watch that all solutions and materials are
RNAse free.
5.2. DNA extraction
After Micro dissection, pipette 50µl Lyses buffer from the Strata Prep PCR Purification
Kit (add 0.05% Proteinase K and 1% Tween 20), or other DNA extraction buffer.
Pre warm the closed tubes in the upright position for 5 min at 42°C then invert the tubes to
contact the buffer with the cells and incubate over night at 42°C
After incubation, spin the tubes 5 min slowly and heat the cap 10 min at 95°C to inactivate the
Proteinase K.
Follow the steps as described in your protocol.