Arcturus
®
HistoGene
®
Immunofluorescence Staining Kit
User Guide
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Information in this document is subject to change without notice.
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TRADEMARKS
The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.
GeneChip is a trademark of Affymetrix, Inc. Cy is a trademark of GE Healthcare. Tissue-Tek is a trademark of Sakura Finetek. RNase AWAY
is a trademark of Molecular Bio-Products, Inc.
© 2010 Life Technologies Corporation. All rights reserved.
12653-00 Rev. C
07/2010
Table of Contents
I. Introduction
A. Background 3
B. Storage and Stability 4
C. Safety Data Sheets
4
D. Related Products
5
II. Kit Components
A. Reagents and Supplies in Kit 6
B. Additional Lab Equipment and Materials Required 6
III. Preliminary Steps
A. Material and Protocol Review 8
B. Recommendations for RNase-free Technique 8
C. Checking RNA Quality in Starting Material 8
IV. Protocol
A. Specimen Freezing 9
B. Slide Preparation 9
C. Primary Biotinylated Antibody Solution Preparation 11
D. Cy3 Streptavidin Conjugate Preparation 12
E. HistoGene LCM Immunofluorescence Staining Protocol 12
V. Troubleshooting
A. Targeted Cells Do Not Lift From the Slide During LCM 14
B. No Staining 14
C. Background Staining 15
D. RNA Cannot be Recovered from the Sample 15
VI. Appendices
A. Tissue Scrape Protocol for Verifying RNA Quality Using 16
the PicoPure RNA Isolation Kit
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3
I. Introduction
A. Background
The HistoGene
®
LCM Immunofluorescence Staining Kit is
the only kit designed to enable retrieval of high-quality RNA
from immunofluorescently stained frozen tissue. It enables
convenient and reliable staining, dehydration and Laser Capture
Microdissection (LCM) of tissue sections with protocols
streamlined and optimized both for optimal LCM captures
and maintaining RNA quality for downstream applications
that require intact RNA, like microarray analysis and RT-PCR.
The HistoGene Immunofluorescence Staining Kit uses a two-
step immunostaining technique that works with any biotinylated
monoclonal antibody. It is based on use of biotin-avidin system:
a primary biotinylated antibody and Cy®3-conjugated
Streptavidin. Avidin is an egg-white derived glycoprotein with
an extraordinarily high affinity for biotin. Many biotin molecules
can be coupled to a protein, enabling the biotinylated protein to
bind more than one molecule of avidin. Cy3 dye is used because it
offers the best staining intensity and works well with the biotin-
avidin system.
The advantages of using the biotin-avidin system are that it
improves sensitivity and reduces background fluorescence.
Furthermore, the high affinity between biotin and avidin assures
the user of a rapidly formed and stable complex between primary
antibody and the avidin-fluorophore conjugate. Thus, the
biotin-avidin system provides highly specific staining with very
little background and enables short staining times necessary to
maintain RNA integrity.
The unique process is performed at 4°C using specially
formulated Staining Buffer. This dramatically increases RNA
yield from captured cells. The HistoGene Cold Block (HIS0101)
makes the 4°C processing very convenient. It holds up to four
slides and has places for reagent tubes. It is designed for use with
the CoolSafe triple density polystyrene cooler (Cat. # CSF-BOX)
Introduction
Liver, kidney and some lymphoid
tissues contain endogenous biotin and
are therefore not recommended for
staining with Streptavidin based IHC
procedures.
This kit should not be used with
antibodies requiring enzyme digestion
or target retrieval.
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4
and –10°C cool brick (Cat. # BRIK-1520) from Diversified
Biotech (www.divbio.com). The proprietary Staining Buffer
perserves the quality of RNA in the tissue sections and preserves
good, specific staining intensity.
The HistoGene Immunofluorescence Kit requires starting with
unfixed, frozen tissues known to contain intact RNA. Thus, we
recommend that you verify RNA quality before you begin. See
Appendix A. All Kit components used are premium grade,
LCM certified and nuclease free to ensure RNA quality is
maintained during the process.
B. Storage and Stability
1. HistoGene LCM Immunofluorescence Staining Kit
Store the staining reagents as follows:
Buffer A (–70°C)
Buffer B (4°C)
Cy3 Streptavidin (4°C)
2. HistoGene Cold Block
Store at 4°C.
C. Safety Data Sheets
Safety Data Sheets (SDS) for kit chemical
components are available from Technical Services.
Call: 1 800 831-6844 option 5.
Do not use on fixed frozen or
formalin-fixed, paraffin-embedded
sections.
Store slides at room temperature and the Cold Block at
4°C. Do not freeze and thaw Buffer A repeatedly.
You can also obtain these sheets from:
www.appliedbiosystems.com.
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5
Introduction
D. Related Products
PicoPure
®
RNA Isolation Kit
For extraction and isolation of total RNA from small samples
particularly Laser Capture Microdissected (LCM) cells. The
PicoPure RNA Kit comes with optimized buffers, MiraCol
Purification Columns and an easy-to-use protocol to maximize
recovery of high-quality total cellular RNA ready for amplification
with the RiboAmp OA 1 Round RNA Amplification Kit.
RiboAmp
®
PLUS RNA Amplification Kit
The RiboAmp PLUS RNA Amplification Kit enables the pro-
duction of microgram quantities of antisense RNA (aRNA)
from nanogram quantities of total cellular RNA. Amplified
RNA produced using the kit is suitable for labeling and use for
probing expression microarrays. The kit achieves amplifications
of up to 1000-fold in one round of amplification, and amplifi-
cations of up to 1,000,000-fold in two rounds. The RiboAmp
PLUS Kit comes with all necessary enzymes, reagents, and
MiraCol purification columns needed to complete the included
amplification protocol.
RiboAmp
®
HS RNA Amplification Kit
The RiboAmp HS RNA Amplification Kit starts with picogram
total cellular RNA input and enables the production of microgram
quantities of antisense RNA (aRNA). The Kit provides the greatest
level of sensitivity in starting RNA quantities to produce enough
RNA suitable for labeling and hybridizing onto expression
microarrays. The RiboAmp HS Kit comes with all necessary
enzymes, reagents, and MiraCol Purification Columns needed
to complete the included amplification protocol.
PicoPure
®
DNA Extraction Kit
The PicoPure DNA Extraction Kit is optimized to maximize the
recovery of genomic DNA from 10 or more cells captured by
LCM. The kit comes with reagents and protocol tested to ensure
complete extraction of DNA from LCM samples prepared with
any standard tissue preparation procedure. DNA prepared using
the kit is PCR-ready and needs no additional purification to perform
amplification.
HistoGene
®
LCM Frozen Section Staining Kit
The HistoGene LCM Frozen Section Staining Kit is used to
process tissue sections for LCM that maximizes the quality and
yield of RNA from LCM cells. The Kit comes with all dehydration
and staining reagents, disposable staining jars, specially treated
slides, and detailed protocol and troubleshooting guide.
Paradise
®
PLUS Reagent System
The Paradise PLUS Reagent System is the only reagent system de-
signed to enable gene expression studies using formalin-fixed paraf-
fin-embedded (FFPE) tissue samples. Components include sample
preparation and staining reagents, RNA extraction and isolation
reagents, RNA amplification reagents and a comprehensive user
guide.
6
II. Kit Components
A. Reagents and Supplies in Kits
The HistoGene Immunofluorescence Staining Kit includes
reagents to stain 32 tissue sections. Fixation and dehydration
reagents are not supplied with the Kit. See additional materials
required below.
Buffer A (8 x 500 µl)
Buffer B (60 ml)
Cy3 Streptavidin (60 µl)
Slides (72)
User Guide
B. Additional Equipment and Materials
Required
1. Materials
PAP pen or ImmEdge pen (Vector Laboratories,
Cat. # H-4000)
RNase-free pipet tips
15 ml nuclease-free Falcon tubes
Microcentrifuge tubes, nuclease-free
RNaseAway (Life Technologies, Cat. # 10328-011)
Kimwipes
Tissue-Tek
OCT Compound (VWR Cat. # 25608-930)
Staining jars (Evergreen Scientific # 222-5450-G8S)
Dry ice
Disposable gloves
Tissue-Tek
Cryomold (VWR Cat. # 25608-916)
2. Equipment
-70ºC freezer
Fume hood
Cryostat with disposable microtome blades
Pipettors: 1000 µl, 200 µl, 100 µl, 10 µl
Cover glass forceps
HistoGene Cold Block (Applied Biosystems Cat. # HIS0101)
Thermometer, celsius
Microslide box, plastic (VWR Cat. # 48444-004)
Refrigerator
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Kit Components
2. Reagents
Acetone
(VWR Cat. # AX-125-4; Fisher Cat. # HC-300-GAL)
Xylene
(VWR Cat. # EM-XX0060-4; Fisher Cat. # HC-700-GAL)
Ethanol 100%
(VWR Cat. # 34172-020; Fisher Cat. # HC-800-GAL)
Ethanol 95%
(VWR Cat. # 34172-022; Fisher Cat. # HC-1100-GAL)
Ethanol 70%
(VWR Cat. # 34172-000; Fisher Cat. # HC-1000-GAL)
Isopentane or 2-Methylbutane (VWR Cat. #JTQ223-7)
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III.Preliminary Steps
A. Material and Protocol Review
This protocol is only for immunofluorescent staining of acetone
fixed frozen tissue sections.
To get the most from your HistoGene LCM Immunofluorescence
Staining Kit, take a few moments to examine the components of
the kit and read through the information in this Section and
Section IV.
B. Recommendations for RNase-free Technique
RNase contamination will cause experimental failure. Minimize
RNase contamination by adhering to the following
recommendations throughout your experiment:
• Wear disposable gloves and change them frequently.
• Use RNase-free solutions, glassware and plasticware.
• Do not re-purify HistoGene LCM Immunofluorescence
Staining Kit components. They are certified Nuclease Free.
• Wash scalpels, tweezers and forceps with detergent and bake
at 210°C for four hours before use.
• Use RNase AWAY
®
solution (Life Technologies) according
to the manufacturer’s instructions on any surfaces that may come
in contact with the sample.
C. Checking RNA Quality in Starting Material
Before you begin preparing specimens, it is critical that you first
check the quality of the RNA in the tissue to ensure that your
samples contain intact, high-quality RNA. See Appendix A.
Preliminary Steps
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IV. Protocol
A. Specimen Freezing
Note that isopentane has a very low flash point and should be
kept away from open flames. Perform procedure in a fume
hood or a well-ventilated space.
1. Place dry ice in an appropriate container.
2. Slowly pour isopentane into container with dry ice, filling
until the isopentane level is just above the layer of dry ice.
3. Bubbling of the isopentane will occur upon its addition to
the dry ice, once this has subsided the isopentane is ready
for use.
4. If necessary, identify specimen on cryomold using a sharpie
pen.
5. Take cryomold and place a thin layer of OCT on the bottom
of it.
6. Collect dissected tissue specimen and place tissue in desired
orientation onto the layer of OCT in the cryomold.
7. Carefully add more OCT until specimen is completely
covered and the cryomold is filled.
8. Carefully place prepared cryomold into the cooled
isopentane.
9. Wait for OCT to completely solidify. If freezing down
additional specimens, the processed specimens can be held
in a separate container with dry ice only.
10. Store frozen specimen in the cryomold in a –70°C freezer
or proceed to slide preparation.
B. Slide Preparation
1. Pre-cool the cryostat to the temperature recommended by
the manufacturer for the specimen you are preparing.
Wear clean disposable gloves
throughout the Specimen Freezing
procedure. Use clean RNase-free
instruments.
For best RNA preservation, freeze
tissue specimens immediately after
dissection.
Wear clean disposable gloves
throughout the Slide Preparation
procedure.
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It is okay to stop at this point in the protocol.
2. Remove and discard old microtome blade. Wipe down the
knife holder and antiroll plate in the cryostat with 100%
ethanol to avoid sample cross-contamination. Dry wet
surfaces with another dry Kimwipe.
3. Install a new disposable microtome blade in the cryostat.
4. Set cutting thickness to 8-10 µm.
5. Place a microslide box on dry ice near the cryostat.
6. Transfer the cryomold containing your specimen from
the –70°C freezer to the cryostat, transporting on dry ice if
necessary.
7. Wait a minimum of 10 minutes for your specimen to
equilibrate with the temperature of the cryostat.
8. Mount specimen to specimen holder with OCT. Cut 8-
10µm sections.
9. Mount sections toward the center of a room temperature
LCM microslide. Place slide immediately into microslide
box on dry ice. Do not allow slide to dry at room temperature.
10. Discard slides with folded or wrinkled sections.
11. If you are cutting more than one specimen, use a new unused
section of the disposable microtome blade or a new blade for
each one. In addition, wipe down the knife holder and anti-
roll plate with 100% ethanol in between each specimen to
avoid cross-contamination.
12. Upon completion of sectioning, the surface of the specimen
must be protected with a thin layer of OCT prior to storing
at –70C. This can be accomplished by applying a small
amount of OCT on the surface of the block and immediately
applying a metal block that has been pre-cooled to the
cryostat’s temperature. To separate the two objects once the
OCT has solidified, take a razor blade and place it between
the metal block and the OCT. Once separated, carefully
remove the specimen block from the specimen holder using
the razor blade. Remove any excess OCT surrounding the
block with the razor blade and replace the specimen into its
cryomold.
13. Proceed immediately to the “Staining and Dehydration”
segment of the protocol or store slides in a slide box at –70°C
for up to two months.
Protocol
Frequent cycling of the tissue block
from –70
o
C to –20
o
C for cryosectioning
may accelerate RNA degradation. For
best results, cut and mount a sufficient
number of sections for two months’
use during one cryosectioning session.
Store the mounted sections at –70
o
C
until needed.
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C. Primary Biotinylated Antibody Solution Preparation
When using the HistoGene LCM Immunofluorescence Staining
Kit, it is important to use primary antibodies proven to be of
high quality. They should be tested in a conventional
immunostaining procedure and shown to produce good staining
intensity with low background before proceeding with the
HistoGene LCM Immunofluorescence Staining protocol.
The recommended concentration of the primary biotinylated
antibody should be between 30–100 µg/ml. The final
concentration of the primary antibody should be optimized
by the user by performing a serial dilution staining experiment
using the HistoGene Immunofluorescence Kit’s Staining Buffer
(Buffers A & B combined).
Biotinylated antibodies against many different antigens are
now commercially available. If a certain antibody cannot be
obtained from a commercial source the biotinylation can easily
be performed using a biotinylation kit (Pierce cat # 21338,
Sigma Cat. # B-TAG).
It should be noted, that some commercially available
biotinylated antibodies have low IgG concentrations and need
to be used undiluted.
To perform a negative control staining, use a biotinylated control
antibody from the same animal species and of the same isotype
as your primary antibody. Dilute to the same working
concentration as the primary antibody. (Mouse control antibodies
can be obtained from DAKO Cat. # X0928).
Working solution of the primary biotinylated antibody and the
negative control antibody should be prepared in Staining Buffer
(Buffers A & B) and held at 4°C. The recommended
concentration of Buffer A in all antibody solutions is 10 mM. If
preparing the antibody solution dilutes the Buffer A
concentration below 10 mM, add an adequate volume of the
Buffer A stock solution (100 mM) to the antibody solution.
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D. Cy3 Streptavidin Conjugate Preparation
Working solution of Cy3 Streptavidin should be prepared in
Staining Buffer (Buffer A & B) and held at 4°C, protected
from light. The recommended dilution of the Cy3 Streptavidin
is 1:100. User should, however, determine the optimal staining
concentration of the Cy3 Streptavidin conjugate by performing
a serial dilution staining experiment using the HistoGene
Immunofluorescence Kit’s Staining Buffer (Buffers A & B).
E. HistoGene LCM Immunofluorescence Staining Protocol
Minimizing the time required and keeping the samples cold
during immunoflourescent staining is critical for obtaining high
RNA quality and yield. For best results, the staining protocol
should be performed within a maximum of 10 minutes, not
including dehydration steps. This requires maintaining a steady
workflow. To help minimize preparation time and keep the
samples cold, we recommend that you do not process more than
4 slides at once. Also do not allow the sections to dry after Step
14 of the protocol. Drying may result in non-specific staining,
loss of specific staining and it may compromise RNA quality.
1. Pre-cool the HistoGene Cold Block in a refrigerator to 4°C.
2. Take out one aliquot of frozen Buffer A and thaw.
3. Prepare fixative and dehydration reagents in the fume hood.
4. Label 5 plastic jars as follows:
a Acetone
b. 70% ethanol
c. 95% ethanol
d. 100% ethanol
e. Xylene
5. Fill each labeled jar with 25 ml of solution.
6. Cool Acetone to 4°C.
7. Take the cold block out of the refrigerator and place it in the
CoolSafe box or on wet ice. Just prior to staining, check the
temperature of the cold block by placing a thermometer in
one of the tube holes. The temperatures should be 4°C or
Protocol
Discard any remaining Buffer A once
thawed.
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The stock solution of Buffer A is
100 mM, and the working solution
should be 10 mM.
Staining procedure is performed at
+4°C by keeping the slides on a cold
block throughout all the steps of the
procedure.
All solutions must be prepared just
prior to staining.
below.
8. Prepare Staining Buffer by adding one aliquot (500 µl) of
the Buffer A into 4500 µl of Buffer B. Store tube in the cold
block. Discard any remaining Buffer A once thawed.
9. Prepare primary antibody solutions as described in Section
IV.C and keep them in the cold block.
10. Prepare the Cy3 Streptavidin conjugate and keep it in the
dark and at 4°C as described in Section IV.D.
11. Remove a maximum of four slides from the –70°C freezer,
allow no more than 30 seconds for the condensation to
disappear prior to fixing in acetone.
12. Place slides in cold acetone (4°C) for 2 minutes.
13. Remove from acetone and air dry the slides in the fume
hood for 30 seconds.
14. Use hydrophobic barrier pen to circumscribe each section
to keep the staining reagents localized on the tissue section.
15. Place all the slides on a cold block.
16. Apply 200 µl of Staining Buffer per section to each slide.
17. Drain off staining buffer and wipe the slide gently, without
touching the section, with a clean Kimwipe.
18. Place the slides on the cold block and apply 100 µl per
section of the diluted primary antibody and incubate for 3
minutes.
19. Drain off the antibody solution and rinse the sections by
applying 200 µl of Staining Buffer per section, drain and
repeat rinsing.
20. Drain off staining buffer and wipe the slide gently with a
clean Kimwipe.
21. Place the slides on the cold block and apply 100 µl per
section of the diluted Cy3 Streptavidin and incubate for 1
minute.
22. Drain off and rinse by applying 200 µl of Staining Buffer
per section, drain and repeat rinsing.
23. Immediately proceed to the dehydration protocol.
Dehydration is performed at room temperature.
Do not allow the sections to dry. Work
with one slide at a time through steps
#20 and 21.
Discard any unused Staining Buffer
and diluted antibody solutions.
Do not allow the sections to dry. Work
with one slide at a time through steps
#17 and 18.
Place the slide back on the cold block
if processing multiple slides.
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Process only the number of slides
that you will be able to microdissect
within 2 hours after the final
dehydration step. The RNA in the
processed sample degrades even after
final dehydration step!
V. Troubleshooting
1. Place the slides in 70% ethanol for 30 seconds.
2. Transfer into 95% ethanol for 30 seconds.
3. Transfer into freshly dispensed 100% ethanol for 30
seconds.
4. Transfer into xylene for 5 minutes.
5. Dry the slides in a fume hood for 5 minutes.
6. Immediately perform Laser Capture Microdissection.
A. Targeted Cells Do Not Lift From the Slide
During LCM
1. The sample may contain residual water. Ensure that the
ethanol solutions are fresh. Ethanol is hygroscopic. Keep
the ethanol bottles tightly capped, and do not pour ethanol
solutions until you are ready to use them. If you suspect that
the 100% ethanol solution has absorbed water, purchase a
new bottle.
2. The sample may have dried between protocol steps. Perform
all steps of the protocol at a steady pace.
B. No Staining
1. Make sure that the primary antibody is used at right
concentration and that it is active. Perform a titration
experiment using both high and low concentration of the
primary antibody.
2. You can test the activity of the antibody on a section taken
from another known positive tissue.
3. Make sure that the Cy3 Streptavidin is not inappropriately
diluted. Perform a titration experiment using both high and
low dilutions of the Cy3 conjugate.
Troubleshooting
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C. Background Staining
1. The primary antibody used is too concentrated. Perform a
titration experiment using both high and low concentrations
of the primary antibody to achieve clean specific staining
without background.
2. The primary antibody may cross-react with other tissue
epitopes and bind non-specifically. Change source or species
of the primary antibody.
3. Make sure that sections are kept moist during all steps of
the staining.
4. Endogenous biotin may be present in the tissue. To check if
this is occurring perform a staining omitting the primary
antibody application.
5. Tissue such as kidney, liver and adrenals as well as some
lymphoid tissues contain different levels of endogenous
biotin and should not be used with a biotin/avidin system.
D. RNA Cannot be Recovered from the
Sample
1. The sample starting material may contain poor quality RNA.
Freeze the sample immediately following dissection, and
take care to use RNase-free technique.
2. RNA may become degraded during RNA isolation. Wear
gloves; use RNase-free technique and RNase-free
instruments and reagents. Wipe down the Laser Capture
Microdissection System with RNase AWAY prior to use.
3. RNA may not be fully extracted and isolated from cells on
the LCM cap. Use the Arcturus PicoPure RNA Isolation
Kit or another guanidinium extraction method. Perform
RNA extraction immediately after LCM to ensure complete
extraction and optimum recovery of RNA.
4. The starting material quantity may be insufficient. Use at
least 500 captured cells.
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VI. Appendix
Appendix
A. Tissue Scrape Protocol for Verifying RNA
Quality Using the PicoPure RNA Isolation Kit
Applied Biosystems recommends verifying the integrity of RNA in
the tissue sample before proceeding with staining and Laser
CaptureMicrodissection (LCM) procedures. This enables
you to understand the quality of the RNA in the experimental
sample before proceeding with further downstream processing.
This protocol is recommended for all new frozen tissue samples.
The protocol involves preparing and dehydrating a tissue section,
then scraping the entire tissue section into a 0.5 ml tube. RNA is
then extracted from the sample using a modified version of the
PicoPure
®
RNA Isolation Kit (Catalog # KIT0204) protocol for
larger amounts of tissue. Finally, the Lab-on-a-Chip System
(Agilent) or a gel can be used to assess 28S and 18S ribosomal
RNA integrity. If ribosomal bands are detected, then the sample
contains viable RNA and is therefore a good candidate for LCM.
If the ribosomal RNA bands are faint or not present, then the
sample may contain degraded RNA.
For more information, visit: www.appliedbiosystems.com, or
call technical support at: 1 800 831-6844 option 5.
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