Histology Applications and Laser Microdissection - Genome ...

Histology Applications
and Laser Microdissection
Mari Ann Mailhiot BA HTASCP
Application Specialist
Leica Microsystems
mari.ann.mailhiot@leica-microsystems.com
800 248 0123 x7267

Snap Freezing
• In a beaker or specimen container, add crushed or pieces off dry
ice to 2 methyl butane to make a slurry mixture of the two (work in a
hood).
• When bubbling stops, the isopentane is at correct freezing
temperature of approximately –90C.
• Precool isopentane in a beaker surrounded by dryice first. This will
help the isopentane from bubbling over when you add it to the dry
ice.
• Immerse OCT embedded tissue slowly. Eventually it will float to the
bottom of the isopentane.
Note: Please be sure to evaporate isopentane
away after freezing to prevent blowing up of
freezers.

Snap Freezing
• Place liquid nitrogen in a styrofoam container.
• You will need a support rack of some sort to hold a petri
dish lid.
• Place stryofoam container inside petri dish lid.
• Place tissue in a disposable mold, embed in OCT.
• Or place tissue embedded in OCT on a coverslip and
place in the liquid nitrogen.
• No isopentane involved with this method.

Cryosectioning Techniques
• Wedge shaped knife is
commonly used.
• An anti roll device is
designed specifically for
that knife.
• The anti roll keeps
sections from curling up.
• Sometimes a brush is
used to help ease the
section down the knife.
• Keep blank slides in
cryostat.
• This will help with
placement of tissue on
slide.

Basic criteria for consistent
Frozen sectioning
• Sharp knife
• Proper knife alignment
• Knife edge free of defects
• Properly adjusted anti roll
device
• Optimum sectioning
temperature
• Tissue frozen at –80
should warm up to at least
–20 for good sectioning
results.
• Each tissue has a firmness
related to its lipid and
water content e.g. fat
colder, brain warmer.

Obtaining Quality Paraffin
Sections Requires
• A processed and embedded specimen of the highest
quality
• A highly skilled operator
• Use of the proper microtome and knife or disposable
blade
• Impeccable maintenance of the microtome and
knife or disposable blade

Factors Affecting
Specimen Integrity
• Specimen cut off from blood supply
• Chemical & thermal treatment of surgical
site
• Physical trauma
• Freezing and thawing
• Fixation delays or exposure issues

Fixation...
• ... is the most important step
in histotechnology
• …even more so for molecular
analysis:
- paraformaldehyde not
RNA-friendly
- use 70% alcohol

Factors Affecting the
Specimen During processing
• Use of heat, vacuum/pressure
• Types of reagents and their strength
• Number of steps
• Duration of exposure
• Cleanliness of reagents
• Use of sponges, paper & biopsy cassettes

Dehydration
The key to proper dehydration is to
expose the specimen long enough to
remove the free water molecules
but
not so long that the bound water is
removed from the tissue.

Paraffin as an Infiltration
Medium
• Keep 2° to 4° above the melting point
• Last station must be free of contamination
• Enhanced by vacuum, but use caution with small,
fragile biopsies
• Limit exposure to reduce shrinking and hardening
of tissue

Selection of Embedding
Paraffin
• Low m.p. paraffin is usually softer
‣ Sectioning can be difficult, ribboning is easier
• High m.p. paraffin is usually harder
‣ Better support for hard tissue, thinner sections
possible but ribboning is more difficult

Deparaffinization of Slides
• Using xylene - Foil slides cannot remain in xylene
longer than 1 minutes
• Using a xylene substitute - Foil slides cannot
remain longer than 1 minutes
• When working with xylene substitutes remember
that they have zero tollerance for water

H&E Staining Paraffin
Sections
• Xylene 3 changes 20 secs. each
• Absolute Alcohol 2 changes 10 dips each
• 95% Alcohol 2 changes 10 dips each
• Tap Water – until water run off slides evenly
• Hematoxylin Progressive Mayer or Harris 10
minutes

H&E Staining
• Running Tap Water – until most of the stain is off
• Acetic Acid or Clarifier – 1 to 3 minutes
• Running Tap Water
• Ammonia Water 0.25% or lithium carbonate 0.5% -
until blue
• Running tap water- 1 min
• Eosin or eosin-phloxine - 1 to 3 min
• 95% Alcohol, two changes - 10 to 15 dips each
• Absolute Alcohol, three changes 10 - 15 dips

H&E Staining
for Frozen Sections
• Place a slide in 70% alcohol about 1 minute
• Rinse with water until slide looks clear
• Then stain in hematoxylin and eosin
* Note: OCT interferes with laser microdissection,
and need to be removed completely. 70% alcohol
will clear the OCT compound. Keep in 70%
longer for thick sections.

Toluidine Blue
• Toluidine blue 0.1 g
• Dist Water 100 ml
• Deparaffinize slides
and hydrate to distilled
water
• Stain sections in
toluidine blue 10 min
• Rinse in distilled water
• Quickly dehydrate
through 95% and
absolute alcohol.

Methyl Green – Pyronin Y
Stain
Solution A – 0.2M acetic acid
• Glacial Acid Acid 2.3 ml
• Dist. water
197.7 ml
Solution B – 0.2M sodium acetate
• Sodium acetate trihydrate 2.72 g
• Dist. Water 100 ml

Methyl Green – Pyronin Y
Stain
• Acetate Buffer Solution Working
• Solution A
150 ml
• Solution B
50 ml
Adjust the ph to 4.2 with sodium hydroxide or
acetic acid

Methyl Green – Pyronin Y
Stain
• Purification of Methyl Green
• Place solution in separatory funnel.
• Add 50 ml of chloroform and shake.
• Allow methyl green and chloroform to separate
into layers.
• Dicard chloroform – bottom layer.
• Repeat until the chloroform is clear and all traces
of methyl violet have disappeared.

Methyl Green – Pyronin Y
Stain
• Allow the methyl green staining solution to stand
in an open flask overnight so that any residual
chloroform evaporates.
• DNA
• RNA
Green to blue green
Red

Methyl Green – Pyronin Y
Stain
• Purified methyl green solution 10 ml
• Pyronin Y 10 mg (0.01g)
• Dye source methyl green CL 42585
• Dye source pyronin G (Y) CL 45005
• Note: Please do not substitute any other dye index

Methyl Green – Pyronin Y
Stain
• Deparaffinize sections in xylene
• Hydrate in two changes of absolute
• Two changes of 95% alcohol
• Rinse in dist water
• Stain one slide at a time by placing the slide on a
staining rack. Using a pipette flood the slide with
methyl green-pyroninY solution. Stain for 5
minutes.

Methyl Green – Pyronin Y
Stain
• Quickly rinse slide with dist water
• Blot dry
• Dip slide into two changes of acetone (15 quick
dips)

Conclusion
• All solutions should only be used for one patient or
animal and discarded.
• Fresh chemicals and stains per patient or animal.
• Subscribe to the histonet – a lot of help!
Histonet@pathology.swmed.edu

Leica Protocols
Preparation protocols for Leica laser microdissection
1. Preparation of membrane-coated glass slides
2. Applying specimen to slide
3. Staining of sections on membrane-coated slides
3.1 Paraffin-embedded
3.2 Fresh frozen tissue
4. Immunostaining
4.1 Paraffin-embedded tissue
4.1.1 Optional heat-induced epitope retrieval (HIER)
4.1.2 Alkaline Phosphatase Immunolocalization
4.2 Fresh frozen tissue
5. Preparation of microdissectate for DNA analysis
6. Preparation of microdissectate for RNA analysis/ cDNA synthesis
6.1 First strand cDNA synthesis of RNA purified from microdissected tissue
7. PCR
8. Protein-analysis
9. In situ hybridization
10. Cytospin, Cell-Monolayer
11. Blood smear
12. Chromosomes
13. Living cells
1

Leica Protocols
1. Preparation of membrane-coated glass slides
Membrane-coated slides (1mm for 20-40x; 0.17mm for 100x magnification [oil]) can be prepared as follows:
Gloves must be worn at all times.
Conventional microscopic slides are cleaned meticulously with acetone/ethanol and left to dry in a dustfree
environment.
Slides are dipped in distilled water and mounted with appropriately sized membranes
(approx. 21mm x 42mm). Take care of wrinkles.
Make sure to leave at least 2mm uncovered glass surface around the edges.
After complete evaporation of water from the uncovered slide areas the membranes are sealed around
the edges with clear nail varnish and left to dry for at least 1 hour at room temperature (RT).
Optional: Siliconising of glass slides by dipping into dimethyldichlorosilane and drying for 10 min. at 40°C.
Siliconising of glass slides may improve efficiency of cutting/falling for larger samples.
Optional: apply nail varnish only to two sides of the foil and put the slide in an oven at 40 °C overnight.
Then fix the other two sides of the foil with the nail polish. This will improve the drying and make sure that
there is no water between foil and slide.
Leica offers prepared PEN foil slides ready for step 2
Immunohistochemical detection requires use of LEICA membrane-coated slides.
2. Applying specimen to slide
Optional: For reduction of electrostatical charge of the foil and possible destruction of RNases the slide is
incubated for 30 min. in a UV chamber (max. power). UV will improve the fixation of the foil.
Optional (frozen sections): For better attachment of tissue/cells to foil tissue adhesive can be applied as
follows. Apply shortly before use one drop of diluted tissue adhesive (TA, Diagnostic Products
Corporation, van Golsteinlaan26, 7339 GT Apeldoorn, the Netherland) to foil. Spread gently over foil with
pipette tip and remove excess TA. Perform powerful shaking with the arm such that only a thin film of TA
remains on the foil. Allow the TA to dry for 15 min. at 37°C.
Collect tissue section (+/- 5 µm) on the foil. Allow tissue section to dry for 30 min. or 1h. respectively at
room temperature.
2

Leica Protocols
3. Staining of sections on membrane-coated slides
3.1 Paraffin-embedded tissue
Dewax in xylol for 45 seconds (max. 3 minutes) , as otherwise the adhesive fixing the foil to the specimen
slide will dissolve. As an alternative, intermedium substitutes such as Paraclear can be used instead of
xylol.
For rehydration, the sections are immersed for 30 seconds each in absolute ethanol (3x), 96% ethanol (1x),
70 % ethanol (1x) and finally in distilled water.
Nucleus staining with haemalum (Mayer technique) for 5 min.
Rinse in tap water
Differentiate: depending on the recipe of the haemalum used, differentiation must be made in 0.5-1% HCL
alcohol under microscope control (Mayer´s haemalum, prepared with the following recipe: dissolve
1g haematoxylin in 1000ml distilled water, then add, one after the other: 200 mg sodium iodate, 50 g potash
alum, 50 g chloral hydrate and 1 g citric acid, does not require differentiation)
Bleach nuclei blue in tap water for 5 to 10 min. (microscope control)
The staining of the cytoplasm can be done with eosine or erythrosine for 1 min. A 1:1 mixture of the two
solutions is recommended.
Rinse the sections well in distilled water then air- dry. If necessary, it is also possible to differentiate in
70-96 % ethanol and dry after returning to distilled water.
3.2 Fresh frozen tissue
Follow protocol for paraffin sections after rehydration step.
Alternative protocol:
Stain tissue section for 1 min. with haematoxyline or 10 seconds with methyl green or toluidin blue and
rinse in sterile water. There are indications that methyl green or toluidin blue give better PCR results than
haematoxylin. Carefully remove excess water by placing a filter on the sample and gentle striking with a
finger. Allow to dry further for 30 min. at 37°C.
3

Leica Protocols
4. Immunostaining
4.1 Paraffin-embedded tissue
4.1.1 Optional heat-induced epitope retrieval (HIER)
Transfer the rehydrated 3µm paraffin sections into 1000ml HIER citrate pH 6.0 buffer.
10x stock
7,65g Citric acid
48,2g Sodium citrate
water ad 2000ml
Bring to the boil in a pressure cooker for 1-3 minutes, let equilibrate to room temperature and transfer into
TBS pH 7.4 (50 mM Tris, 150 mM NaCl).
4.1.2 Alkaline Phosphatase Immunolocalization
Incubate sections in DAKO Protein Block (No. X0909) to reduce unspecific binding. Incubate the section in
100mµl TBS/0.5 % BSA with the first antibody for 1 h at room temperature or 37°C (alternatively overnight
at 4°C).
Wash section once with TBS-Tween 0.01%, once with TBS.
Incubate the section in 100mµl TBS/0.5 % BSA with the second biotinylated antibody for 30min at room
temperature or 37°C.
Wash section once with TBS-Tween 0.01%, once with TBS.
Incubate the section in 100µl Streptavidin-Alkaline Phosphatase conjugate (ABC-Kit Vector No. AK5000)
for 30 min at 37°C.
Wash section once with TBS-Tween 0.01%, once with TBS.
Develop with Sigma Fast Red chromogen under microscopic control.
Wash section once with distilled water.
Counter stain with Mayer’s Haemalum.
Bleach nuclei blue in tap water for 5 to 10 min. (microscope control)
Coverslip the section in Aquatex.
4.2 Fresh frozen tissue
Frozen tissue is cut at preferably 3-5µm thickness in a cryostat.
Sections are fixed in acetone 0.1% NP40 for 5 min.
Proceed to incubation in DAKO Protein Block (Nr.: X0909) to reduce unspecific binding as above under 4.1.
4

Leica Protocols
5. Preparation of microdissectate for DNA analysis
Microdissectates (frozen or paraffin-embedded) are collected individually or pooled in 20-100µl of lysis
buffer (10 mM Tris-HCl pH 8.0, 1% Tween-20).
After capping the solution is spun for 15 s and 1-5µl of Proteinase K stock solution (e.g.
100mg/ml) is added.
The microdissectate solution is kept at 55°C for a minimum of 60min (preferably o/n).
Proteinase K is heat inactivated 99°C for 10 min.
Use 1-10µl aliquots in subsequent PCR analyses.
Alternatively spin column extraction (QIAGEN DNA blood/tissue kits) can be used.
6. Preparation of microdissectate for RNA analysis/ cDNA synthesis
It is recommended to wear gloves, to work, if not otherwise stated on ice and to use RNase-free tubes and
DEPC-water.
Microdissectates (frozen or paraffin-embedded) are collected individually or pooled in 20-100µl of QIAGEN
RNAeasy Mini Kit (Cat. No. 74 106) LGT/ß-ME buffer dispensed centrally in the lid of an 0.5ml Eppendorf
cup.
After capping the solution is spun for 15 s and transferred into 1.5ml Eppendorf cup.
Follow QIAGEN RNAeasy protocol for further procedures.
Elute into 30µl of distilled water and keep at -80°C
6.1 First strand cDNA synthesis of RNA purified from microdissected tissue
Customary precautions for RNA handling should be observed.
Per reaction the Reverse Transcription Master Mix consists of
4 µl dNTP-Mix; 10 mM each (Applied Biosystems N8080260)
4 µl MgCl2; 25mM
2 µl GeneAmp 10x Buffer II (Applied Biosystems N8080010)
1 µl Random hexamer primers (Applied Biosystems N8080127)
1 µl MuLV reverse transcriptase (Applied Biosystems N8080018)
1 µl RNAse Inhibitor (optional) (Applied Biosystems N8080119)
Distilled water ad 13µl, mix at RT and use up to 8µl of the RNA solution (see 5.) in a 20µl total reaction
volume.
Leave 10min at RT, then move to 42°C for 60min.
Use 2-10µl aliquots in subsequent PCR analyses.
5

Leica Protocols
7. PCR (Polymerase Chain Reaction)
(Mullis et al. 1987, Meth. Enzymol 155, 335-350 ; Sambrook et al. 1989, Molecular cloning: A laboratory
manual, Cold Spring Harbour, New York; Cold Spring Harbour Laboratory Pres
The PCR is used to amplify defined DNA sequences using special oligonucleotid primer. The cyclic
reaction is carried out in three steps: The denaturation of the DNA by heat, the sequence specific
annealing of the primer and the polymerisation by the heat stable (Taq)-polymerase.
For example:
PCR reaction:
1x Taq-buffer complete with 1.5 mM MgCl2
dNTP-mix (à 200µM)
(Taq)-polymerase (1u)
Primer 1 (2 µM)
Primer 2 (2 µM)
Template DNA
A.dest to 50 µl
cycle Denaturing Annealing polymerisation
1 95°C, 2 min.
30 94°C, 30sec. 50°C, 45sec. 72°C, 1min
1 72°C, 5min
An aliquot of the amplified DNA is ready for agarose gel analysis
8. Protein-Analysis
The cells of the section allow also protein analysis, e.g.
SDS-PAGE
westernblotting
2-D gel elektrophoresis.
For example the cells can be directly cutted into SDS-sample buffer (10% (v/v) glycerol, 5% (v/v) 2-
mercaptoethanol, 2% (w/v) SDS, 0,05% (w/v/) Bromphenolblue, 2 mM Tris-base), heated and loaded to a
normal polyacrylamid gel (Laemmli, U.K. 1970, Nature 227, 680-685).
For two-dimensional gel electrophoresis IEF-buffer (e.g. 9.5 M urea, 2% NP40, 5% 2- mercaptoethanol)
(O´Farrell 1975, J Biol Chem, 250, 4007-4021; O´Farrel, Goodman 1977, Cell, 12, 1133-1142; Shoeman,
Schweiger 1982, J Cell Sci, 58, 23-33) can be used.
Westernblot analysis can be carried out following standart procedure (e.g.Towbin, Staehelin, Gordon,
1979, Proc Natl Acad Sci USA, 76, 4350-4354)
9. In situ hybridisation
Standart protocols for in situ hybridisation. The foil is heat stable for more than 120 °C
6

Leica Protocols
10. Cytospins
Spin cells with a centrifuge to the slide using standart protocol. It is also possible to spin cells to Menzel
Superfrost slides without foil.
Human white blood cells:
Cytospin methanol fixed human white blood cells on microdissection slide. A drop of fixed cells is pipetted
into 1 ml of 50% acetic acid (to remove cytoplasmic proteins) contained in a specially designed centrifuge
bucket. After centrifugation, the slide is air dried and Giemsa stained according to the above mentioned
procedure
11. Blood smear
One drop of freshly collected full blood is smeared on a foiled microdissection slide, air dried overnight
and Giemsa stained according to the above protocol.
12. Chromosome preparation
Chromosomes from white blood cells from human peripheral blood
Cells were cultured for 72 hours, then treated with colcemid (to arrest cells in metaphase), then treated
with a hypotonic solution (to let them swell), and finally fixed in methanol/acetic acid (3+1, v/v). Fixed cells
were dropped on the foiled microdissection slides and were air dried overnight. Then they were stained
with Giemsa staining solution (1:20 diluted in Gurr buffer, which is a standard buffer used for Giemsa
staining made by dissolving commercially available tablets) for 3 minutes. After washing in water they
were air dried.
13. Living cells
It is recommended to UV treat the PEN foil for sterilization. Then cells can be grown in culture medium at
the foil. Single cells can be selected by laser cutting.
7

QIAGEN Supplementary Protocol:
Isolation and amplification of DNA from microdissected tissue
samples using HotStarTaq ® DNA Polymerase
This protocol is designed for the isolation and amplification of DNA from microdissected animal
tissue samples using proteinase K (e.g., QIAGEN ® Proteinase K, cat. no. 19131) and HotStarTaq ®
DNA Polymerase (cat. no. 203203).
Introduction
Laser-microdissected tissue specimens present a particular challenge for molecular analysis, as
nucleic acids must be purified from very small amounts of starting material. In addition, fixation
and staining steps may compromise the integrity of DNA, and it may be necessary either to modify
fixation protocols or to use cryosections from flash-frozen specimens to minimize this problem.
A wide range of equipment and consumables for sectioning, staining, and microdissection of
specimens is available from Leica (www.leica-microsystems.com).
In this procedure, if the DNA to be isolated is only to be used for PCR, it is possible to digest the
sample in an appropriate volume of PCR buffer using proteinase K, then inactivate the enzyme and
use aliquots of the mixture for subsequent PCR, as described in Protocol 1 (direct procedure) below.
It is often necessary to clean up the sample prior to PCR, to remove possible PCR inhibitors such as
melanine, spermidine, or eosin. In particular, DNA extracted from formalin-fixed tissues performs
much better in PCR following cleanup, as described in Protocol 2 (indirect procedure) below.
Note: Depending on the fixation protocol, the age of the samples, the staining procedure, and the
storage conditions used, the sample DNA may be highly fragmented, thus limiting the size of DNA
fragments isolated.
Protocol 1: Direct procedure
1. Collect the sample directly into a small volume of 1x QIAGEN PCR Buffer. (To obtain
1x QIAGEN PCR Buffer, dilute 10x QIAGEN PCR Buffer with distilled water.)
2. Add 10 µl QIAGEN Proteinase K (i.e., at least 6 mAU) and adjust the volume to 25 µl
with 1x QIAGEN PCR Buffer.
3. Incubate the sample at 55°C for 3 h (16 h for formalin-fixed tissues), with occasional
agitation of the tube. The incubation time may vary depending on the amount of
tissue collected.
Note: When processing formalin-fixed tissue samples, it is necessary to extend the incubation
time in this step to 16–24 h, depending on the fixation time and fixation protocol used.
4. Inactivate the proteinase K by incubating the sample at 95°C for 10 min.
5. Briefly centrifuge the sample to remove drops from the lid of the sample digest tube.
6. To carry out PCR on the whole sample, set up a 50 µl PCR assay by adding 25 µl PCR
master mix containing 1x QIAGEN PCR Buffer, HotStarTaq DNA Polymerase, and a
twofold concentration of primers to the sample digest tube. Alternatively use an
aliquot of the sample digest in a standard PCR reaction.
Amplification of DNA from microdissected tissue samples (PCR02 Aug-02) page 1 of 2

Protocol 2: Indirect procedure
1. Collect the sample directly into a small volume of buffer commonly used for digestion
with proteinase K.
2. Add 10 µl QIAGEN Proteinase K (i.e., at least 6 mAU) and adjust the volume to 25 µl
with the proteinase K digestion buffer.
3. Incubate the sample at 55°C for 3 h (16 h for formalin-fixed tissues), with occasional
agitation of the tube. The incubation time may vary depending on the amount of
tissue collected.
Note: When processing formalin-fixed tissue samples, it is necessary to extend the incubation
time in this step to 16–24 h, depending on the fixation time and fixation protocol used.
4. To efficiently remove any PCR inhibitors and proteinase K, we strongly recommend
cleanup of samples using the QIAquick ® PCR Purification Kit. Follow the QIAquick
PCR Purification Kit Protocol (using a microcentrifuge) as described in the QIAquick
Spin Handbook; and elute the DNA with 30 µl elution buffer. This usually leads to a
much better performance in PCR reactions.
5. Use an aliquot of the eluate in a standard PCR reaction, e.g., using HotStarTaq DNA
Polymerase.
QIAGEN handbooks can be requested from QIAGEN Technical Service or your local QIAGEN distributor.
Selected handbooks can be downloaded from www.qiagen.com/literature/handbooks/default.asp.
Material safety data sheets (MSDS) for any QIAGEN product can be downloaded from www.qiagen.com/ts/msds.asp.
Trademarks: QIAGEN ® , QIAquick ® , HotStarTaq ® (QIAGEN).
Purchase of QIAGEN products for PCR is accompanied by a limited license to use them in the Polymerase Chain Reaction
(PCR) process for research and development activities in conjunction with a thermal cycler whose use in the automated
performance of the PCR process is covered by the up-front license fee, either by payment to Applied Biosystems or as
purchased, i.e. an authorized thermal cycler. The PCR process is covered by U.S. Patents 4,683,195 and 4,683,202 and
foreign equivalents owned by Hoffmann-La Roche AG.
© 2002 QIAGEN, all rights reserved.
Amplification of DNA from microdissected tissue samples (PCR02 Aug-02) page 2 of 2

QIAGEN Supplementary Protocol:
Isolation of RNA from microdissected tissue samples using
RNeasy ® Mini Kits
This protocol is designed for the isolation of RNA from microdissected animal tissue samples using
the RNeasy ® Mini Kit or RNeasy Protect Mini Kit.
Please be sure to read the RNeasy Mini Handbook carefully before beginning this procedure and
especially the detailed RNeasy Mini Protocol for Isolation of Total RNA from Animal Tissues (for
Protocol 1, below) or the RNeasy Mini Protocol for Isolation of Total RNA from Heart, Muscle, and
Skin Tissue, in the handbook appendices (for Protocol 2, below).
Introduction
Laser-microdissected tissue specimens present a particular challenge for molecular analysis, as
nucleic acids must be purified from very small amounts of starting material. In addition, fixation
and staining steps may compromise the integrity of RNA, and it may be necessary either to modify
fixation protocols or to use cryosections from flash-frozen specimens to minimize this problem.
A wide range of equipment and consumables for sectioning, staining, and microdissection of
specimens is available from Leica (www.leica-microsystems.com).
Use of the RNeasy System enables efficient recovery of highly pure RNA. Here we provide
guidelines for use of the RNeasy System for isolation of RNA from cryosections and formalin-fixed
tissue sections, with an additional proteinase K digestion step recommended for the latter.
Important notes before starting
• To minimize RNA degradation, avoid prolonged storage of samples at room temperature
prior to fixation, stabilization in RNAlater RNA Stabilization Reagent,* or flash-freezing in
liquid nitrogen.
• Proteinase K (e.g., QIAGEN Proteinase K, cat. no. 19131) and the RNase-Free DNase Set
(from QIAGEN, cat. no. 79254) are required for Protocol 2, below.
* A modified protocol for preparation of RNAlater preserved tissues for histological studies is
available from QIAGEN Technical Services; please inquire.
Isolation of RNA from microdissected tissue samples (RY13 Jul-02) page 1 of 3

Protocol 1: Isolation of RNA from microdissected cryosections
1. Collect the sample directly into an appropriate volume of Buffer RLT (the volume
depends on the collection vessel used for the microdissection, but should not be
greater than 350 µl).
2. If necessary, transfer the sample and Buffer RLT into a larger reaction vessel (such as
a 1.5 ml or 2.0 ml microcentrifuge tube).
3. Adjust the sample to a final volume of 350 µl Buffer RLT
4. Vortex the sample for 30 s.
No further homogenization steps are necessary.
Note: We recommend adding 20 ng of carrier RNA to the cell lysate before loading it onto
the RNeasy spin column membrane. The carrier RNA will co-purify with the RNA from the
cells, so make sure that this will not interfere with any downstream analyses, such as RT-PCR.
Nearly any carrier RNA can be used, except for tRNA and other RNAs

Protocol 2: Isolation of RNA from microdissected formalin-fixed
tissues
Note: Depending on the fixation protocol, the age of the samples, the staining procedure, and the
storage conditions used, RNA can be highly fragmented into pieces smaller than 300 nucleotides,
thus limiting the size of RNA fragments isolated. Furthermore, as the RNeasy procedure removes
RNA smaller than 200 nucleotides, this can lead to an overall loss in yield if the RNA is highly
degraded.
1. Collect the sample directly into an appropriate volume of Buffer RLT (the volume
depends on the collection vessel used for the microdissection, but should not be
greater than 350 µl).
2. If necessary, transfer the sample and Buffer RLT into a larger reaction vessel (such as
a 1.5 ml or 2.0 ml microcentrifuge tube).
3. Adjust the sample to a final volume of 350 µl Buffer RLT
4. Continue with the RNeasy Mini Protocol for Isolation of Total RNA from Heart,
Muscle, and Skin Tissue in the RNeasy Mini Handbook (3 rd edition) appendices, from
step 5.
Note: This protocol has not been tested with the omission of the optional DNase treatment.
We recommend adding 20 ng of carrier RNA to the cell lysate before loading it onto the
RNeasy spin column membrane. The carrier RNA will co-purify with the RNA from the cells, so
make sure that this will not interfere with any downstream analyses, such as RT-PCR. Nearly
any carrier RNA can be used, except for tRNA and other RNAs

Leica Microsystems AS LMD
Staining protocol AS LMD
Cresyl violet staining
- Dissolve 1g Cresylviolet in 200 ml H 2 O dest.
- Add 1 ml 10% acetic acid
- Boil until complete dissolution
- Add H 2 O to 250 ml end volume
⇒
Filter the solution before using!!!
Staining
1. Dry sections before staining
2. Fix at 4°C for 30'' in 100% Aceton
3. Wash in 1x PBS for 30''
4. Incubate 30'' in Cresylviolett
5. Wash in 1x PBS for 30''
6. Wash in H 2 O for 30''
7. Let sections dry at room temperature
Example:
Leica Microsystems Wetzlar GmbH Tel. +49(0)6441/29-2476
Ernst-Leitz-Strasse 17-37 Fax +49(0)6441/29-2255
D-35578 Wetzlar Christian.May@leica-microsystems.com

Leica Microsystems AS LMD
Staining protocol AS LMD
Thionin staining
Buffer for Thionin staining protocol
Dissolve 1,36 g NaAc (Natriumacetat, waterfree) or 2,26 g Natriumaceta.t
Trihydrat in 100 ml H 2 O.
Add H 2 O to 250 ml end volume.
Staining solution
Add 0,5g Thionin powder to 100 ml buffer.
Heat gently until complete dissolution.
Staining
1. Dry sections before staining
2. Incubate 30'' to 1 min in staining solution
3. Wash in H 2 O for 30''
4. Let sections dry at room temperature
Example:
Leica Microsystems Wetzlar GmbH Tel. +49(0)6441/29-2476
Ernst-Leitz-Strasse 17-37 Fax +49(0)6441/29-2255
D-35578 Wetzlar Christian.May@leica-microsystems.com