PTEN DNP Probe
Research Use Only
980-1225
06454216001
25
INTENDED USE
The PTEN DNP Probe is designed to hybridize to an approximately 370 Kb region of chromosome 10 that includes the PTEN gene in formalin-fixed, paraffin-embedded human tissue
specimens following staining on VENTANA BenchMark instruments (BenchMark GX, BenchMark XT and BenchMark ULTRA). This probe does not contain repetitive DNA sequences.
For Research Use Only. Not for use in diagnostic procedures.
Figure 1. PTEN DNP Probe
SUMMARY AND EXPLANATION
PTEN DNP Probe is a dinitrophenyl (DNP) probe that spans approximately 370 Kb of the PTEN region on chromosome 10.
In general, in situ hybridization (ISH) uses labeled probes to detect specific DNA or RNA target sequences in fixed tissue. This is accomplished by heating the tissue and probe solution
to denature nucleic acids. The reaction is then cooled, allowing the labeled nucleic acid probe to hybridize to its endogenous complementary target sequence in the tissue.
The hybridization of the probe to the target sequence is visualized with an indirect detection method that locates the bound probe and generates a signal. The most common
techniques for indirect methods use a secondary antibody directed against the species of primary antibody (anti-hapten) and an enzyme with a corresponding substrate chromogen
system. This combination results in a colored precipitate at the site of specific antibody binding. The ultraView SISH DNP Detection Kit uses the indirect method to visualize specific
antibodies bound to antigens by depositing a black colored precipitate.
PRINCIPLE OF THE PROCEDURE
PTEN DNP Probe is formulated for use with VENTANA ultraView SISH DNP Detection Kit and accessory reagents on BenchMark GX, BenchMark XT and BenchMark ULTRA
instruments.
The ultraView SISH DNP Detection Kit detects dinitrophenyl (DNP) labeled probes bound to target sequences using silver in situ hybridization (SISH) in paraffin embedded tissue
sections. First, the section is hybridized with a DNP-labeled probe, followed by incubation with a rabbit anti-DNP antibody, which binds to the DNP hapten on the probe. A multimer
solution, a goat anti-rabbit secondary antibody with a horseradish peroxidase (HRP) enzyme, is applied to detect the rabbit anti-DNP antibody. The visualization of the bound
secondary antibody is accomplished with enzyme (HRP) catalyzed deposition of silver which produces a black precipitate. Silver ions (Ag+) from the Silver ISH DNP Chromogen A
(Silver A) solution are reduced by hydroquinone from the Silver ISH DNP Chromogen B (Silver B) solution to metallic silver ions (Ag0). This reaction is fueled by the substrate for HRP,
hydrogen peroxide (Silver C). The silver precipitate is deposited in the nuclei and the target sequence is visualized as a black dot, which is readily visualized by light microscopy. Figure
1 illustrates the SISH reaction. The specimen is then counterstained with Hematoxylin II for interpretation by light microscopy.
The staining protocol consists of numerous steps in which reagents are incubated for pre-determined times at specific temperatures. At the end of each incubation step, the BenchMark
GX, BenchMark XT and BenchMark ULTRA instrument washes the sections to remove unbound material and applies a liquid coverslip which minimizes the evaporation of the aqueous
reagents from the slide. Results are interpreted using a light microscope and aid in the differential diagnosis of pathophysiological processes, which may or may not be associated with
positive staining for the probe.
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Black Signal
Silver A
Silver B
Silver C
Rabbit anti-DNP Antibody
HRP
Goat anti-Rabbit Antibody
NO2
DNP
N
H
Target
Sequence
Region
NO2
DNP-labeled Probe
ultraView SISH DNP Detection Kit
Figure 2. Silver ISH Reaction
REAGENT PROVIDED
PTEN DNP Probe contains sufficient reagent for 25 tests.
One 1.25 mL vial of PTEN DNP Probe, contains approximately 60 μg/mL of the probe labeled with DNP formulated in a formamide-based hybridization buffer.
MATERIALS REQUIRED BUT NOT PROVIDED
Staining reagents, such as VENTANA detection kits and ancillary components, including negative and positive tissue control slides, are not provided.
STORAGE AND HANDLING
Store at 2-8°C. Do not freeze.
Light sensitive. Store vial in box when not in use.
Every reagent is expiration dated. When properly stored, the unopened reagent is stable to the date indicated on the label. Do not use this reagent beyond the expiration date.
Probe Preparation
PTEN DNP Probe can be diluted to 30 ug/mL with ISH Probe Diluent (760-4409) and filled into a user-fillable Probe Dispenser. If desired, another DIG labeled probe can be mixed in
the same dispenser to allow for dual color staining. For example, this PTEN DNP Probe can be mixed with the Chromosome 10 DIG Probe (980-1223) in one dispenser. This will allow
for dilution of both probes and dual color staining using one dispenser.
SPECIMEN PREPARATION
Routinely processed, formalin-fixed, paraffin-embedded tissues are suitable for use with this probe when used with BenchMark GX, BenchMark XT and BenchMark ULTRA
instruments. The recommended tissue fixative is 10% neutral buffered formalin (NBF).1 See Table 1 for a list of recommended fixatives and fixatives that are incompatible with this
probe. If you have questions about recommended fixatives, contact your local support representative.
Recommended Fixatives
Incompatible Fixatives
Neutral Buffered Formalin (NBF)
Bouin’s
Zinc Formalin
Alcohol Formalin Acetic Acid (AFA)
Alcoholic Formalin
PREFER
Table 1. Recommended and Incompatible Fixatives
Slides should be stained immediately, as quality of nucleic acid targets in cut tissue sections may diminish over time.
It is recommended that positive and negative controls be run simultaneously with unknown specimens.
Sections thicker than 4 μm may require stronger protease treatment than the recommended condition and may exhibit more nuclear bubbling than thinner sections due to excess
paraffin in the tissue. Nuclear bubbling appears as large or small bubbles or vacuoles in the nuclei. Usually this artifact does not interfere with signal enumeration. However, severe
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cases of nuclear bubbling may distort the nuclei or signals such that enumeration is not possible. These specimens may need to be deparaffinized in xylene and alcohol baths prior to
repeat staining on the instrument, or the user may select the extended deparaffinization option in the staining procedure (see Troubleshooting).
WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.
6.
7.
Warning, Product Contains Formamide. Formamide is toxic by inhalation and moderately toxic by ingestion. It is an irritant to skin, eyes, and mucous membranes and is
absorbed through the skin. It may cause harm to the unborn child. Take precautions when handling reagents. Use disposable gloves and wear suitable protective clothing when
handling suspected carcinogens or toxic materials. If reagents come in contact with sensitive areas, wash with large amounts of water
Ensure that the waste container is empty prior to starting a run on the instrument. If this precaution is not taken, the waste container may overflow and the user risks a slip and
fall.
Materials of human or animal origin should be handled as potentially biohazardous and disposed of with proper precautions.
Avoid contact of reagents with eyes, skin, and mucous membranes. If reagents come in contact with sensitive areas, wash with copious amounts of water. Avoid inhalation of
reagents.
Avoid microbial contamination of reagents as this may produce incorrect results.
Consult local and/or state authorities to determine the recommended method of disposal.
For supplementary safety information, refer to the product Safety Data Sheet and the Symbol and Risk Phrase Guide located at www.ventana.com.
STAINING PROCEDURE / INSTRUCTIONS FOR USE
VENTANA probes have been developed for use on VENTANA BenchMark series of instruments in combination with VENTANA detection kits and accessories. A recommended
staining protocol for the BenchMark ULTRA instrument with the ultraView SISH DNP Detection Kit is listed in Table 2. Staining protocols for the other VENTANA staining platforms will
have to be optimized by the end user.
The parameters for the automated procedures can be displayed, printed and edited according to the procedure in the instrument’s Operator's Manual. Refer to the appropriate
VENTANA detection kit package insert for more details regarding in situ hybridization (ISH) staining procedures.
Use the following staining procedures to perform the PTEN DNP Probe assay on BenchMark ULTRA instruments:
Instrument Platform
Staining Procedure
BenchMark GX
GX Dual Color Open Probe
BenchMark XT
XT Dual Color Open Probe
BenchMark ULTRA
ULTRA Dual Color Open Probe
Staining Condition
Deparaffinization
BenchMark ULTRA
Extended II
69°C
CC2
Cell Conditioning
82°C
3 cycles
Enzyme
ISH Protease 3, 20 minutes
Denature
Probe
Auto
Dispense
Hybridization Incubation Temp and Time
Stringency Wash Incubation time
80°C for 8 minutes
44°C for 6 hours
3 cycles
72°C
8 minutes per cycle
Silver
Detection
–Incubation Time
Silver Chromogen
8 minutes
Red
Detection
–Incubation Time
24 minutes
Red Chromogen
8 minutes
24 minutes
Counterstain
Hematoxylin II for 8 minutes
Post Counterstain
Bluing Reagent for 4 minutes
Table 2. Recommended Staining Conditions for PTEN DNP Probe on BenchMark ULTRA instruments.
Due to variation in tissue fixation and processing, as well as general lab instrument and environmental conditions, it may be necessary to increase or decrease the probe hybridization,
cell conditioning or protease pretreatment based on individual specimens, detection used, and reader preference.
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QUALITY CONTROL PROCEDURES
Positive Control Specimen
Normal PTEN signals (1 to 2 copies per cell) act as internal positive controls and must be visible in the sample using 20X, 40X, and/or 60X objectives. However, not all cells will exhibit
single gene copy due to biological heterogeneity. Specific nuclear staining may be located in various cells including: stromal fibroblasts, endothelial cells, lymphocytes, and nonneoplastic cells. If the positive controls fail to demonstrate positive staining, this may indicate a reagent or instrument problem. Since every specimen has an internal positive control
(i.e., appropriate SISH staining in normal cells), this acts as the true “positive control”.
A laboratory-specific positive specimen control is recommended to be used with every staining procedure performed.. Such controls are useful to monitor all steps of the procedure,
from specimen preparation through staining. Results with the test specimens should be analyzed on the same run.
Positive Reagent Control
A positive reagent control should be run during assay verification and troubleshooting since DNA accessibility may vary depending on fixation method and pretreatment of the
specimen.
Unexplained Discrepancies
Unexplained discrepancies in controls should be referred to your local support representative immediately. See the Troubleshooting section of this insert. Identify and correct the
problem, then repeat the run.
Assay Verification
Prior to initial use of a reagent in a research procedure, the performance of the reagent should be verified by testing it on a series of specimens with known ISH performance
characteristics. These quality control procedures should be repeated for each new lot of reagents, or whenever there is a change in assay parameters.
ANALYSIS OF RESULTS
A qualified reader experienced in the microscopic interpretation of anatomic pathology specimens, ISH procedures and the recognition of single and amplified PTEN copies (which
require microscopic examination using 20X, 40X, and/or 60X objectives) must evaluate controls before interpreting results.
Note: Use of 100X objective is not recommended. All of the design verification testing was done using 20X, 40X, and/or 60X objectives.
The following sections describe how to analyze and score slides. Table 3 illustrates how to count discrete signals.
Definitions
1.
2.
Slide Adequacy. A PTEN slide must satisfy two criteria to be deemed adequate for enumeration; if the slide does not meet these criteria, then it cannot be enumerated and the
result is unsatisfactory.
a.
Internal Positive Control. Normal PTEN signals (1 to 2 copies per cell) act as internal positive controls and must be visible in the sample. This nuclear staining may be
located in various non-neoplastic cells including: stromal fibroblasts, endothelial cells, lymphocytes, and other non-neoplastic cell types.
b.
Neoplastic cells. Using 20X, 40X, and/or 60X objectives, the invasive aspect of the tumor must exhibit an enumerable field of SISH signals.
Target Areas for Signal Enumeration. An acceptable target area within the invasive carcinoma exhibits an enumerable field of SISH signals. Signal enumeration should not be
performed in areas that contain weak SISH signal, compressed or overlapping nuclei, or necrosis. If one target area is deemed inadequate for enumeration, it often is possible to
find other target areas on the same slide that are adequate. This can be determined by the presence of normal cells exhibiting appropriate SISH staining in or adjacent to the
target area.
Signal Visualization and Slide Adequacy
SISH signals are visualized as:
1.
2.
3.
4.
Single Copy. A discrete black dot (SISH) is counted as a single copy of PTEN, respectively. Discrete single dots visualized in the internal, physiologic, same slide positive control
(non-neoplastic) nuclei represent the size of a single copy in invasive carcinoma cells for the SISH (black) signal.
Multiple Copies. Discrete single SISH signals visualized in the internal positive control nuclei represent the size of a single copy PTEN in invasive carcinoma cells. The size of the
single SISH signals is used as a reference to determine the relative number of amplified copies in the cancer nuclei.
Clusters. Presence of multiple overlapping signals in the nuclei that cannot be enumerated.
Overlapping nuclei, nuclei with only one color present, and specimens with non-specific staining should not be enumerated. Any nuclei with overlapping Red ISH and SISH
signals that cannot be discerned should be visualized at higher magnifications to discern the two signals or should not be counted. Nuclei that appear bubbled should not be
counted.
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Do not count if nuclei overlap.
Do not count if no signal is present.
Do not count if only signal of one color is present.
Do not count if signals are outside the nuclei.
Count as 1 black (Probe 1) and 1 red (Probe 2) signal.
Count as 2 black (Probe 1) and 2 red (Probe 2) signals.
Count as 1 black (Probe 1) and 2 red (Probe 2) signals. The black signal
is a “doublet”. Count two adjacent signals of same color only if the
distance between the signals is equal or greater than the diameter of a
single signal.
Count as cluster (Probe 1) and 2 red (Probe 2) signals. Note on scoring
sheet that clusters are present for Probe 1.
A red signal close to a black signal should be counted as one red signal
and one black signal. This may require enumeration at 60x objective to
discern. Therefore, count as 4 black (Probe 1) and 2 red (Probe 2)
signals. If overlapping signals cannot be distinguished, do not count that
nucleus.
Cluster of black dots obscuring red signal(s). Higher magnification (60x)
may be utilized in attempts to confirm presence or absence of red
signal(s); otherwise do not count: always count nuclei with clear red
signals. Note the presence of SISH clusters on the score sheet. Nuclei
with visible and higher numbers of red signal should be scored in nuclei
with SISH clusters.
If background SISH “dust” occurs in the nuclei, only count if specific SISH
signals are clearly distinguishable from background.
Pink haze may be observed and should not be mistaken for signal. Red
signal may vary in intensity but is always discrete. The image shows 2
discrete red (Probe 2) signals and 2 black (Probe 1) signals.
Table 3. Examples of Signal Visualization for Dual Stain. This guide can be used for a single or dual color stained slide.
Controls
Normal cells within, or adjacent to, the enumerable target area serve as internal controls of the staining run. Normal cell nuclei should contain an average of 1 to 2 discrete black dots,
indicating that the PTEN has hybridized to its gene. If dual color is run, then the normal cell nuclei should contain an average of 1 to 2 discrete black dots. Failure to detect single gene
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copy in normal cells on any slide on the run indicates that the particular slide is inadequate for enumeration. Using positive control samples or xenograft slides will aid in
troubleshooting potential instrument and/or reagent problems.
LIMITATIONS
General Limitations
1.
2.
3.
4.
Tissue staining is dependent on the handling and processing of the tissue prior to staining. Improper fixation, freezing, thawing, washing, drying, heating, sectioning, or
contamination with other tissues or fluids may produce artifacts, antibody trapping, or false negative results. Inconsistent results may be a consequence of variations in fixation
and embedding methods, or inherent irregularities within the tissue.
Excessive or incomplete counterstaining may compromise proper interpretation of results.
Due to variations in specimen processing it may be necessary to either increase or decrease the ISH protease treatment time or to use a different ISH protease on individual
specimens.
Reagents may demonstrate unexpected reactions in previously untested tissues. The possibility of unexpected reactions even in tested tissue groups cannot be completely
eliminated because of biological variability of tissues. Contact your local support representative with documented unexpected reactions.
Specific Limitations
1.
2.
3.
4.
Bouin’s fixative has been demonstrated to be incompatible with ISH probes. Samples fixed using Bouin’s fixative failed to yield single copy gene detection.
AFA is not a recommended fixative to be used with this assay.
Zinc formalin did not stain as robustly as tissue fixed in either 10% NBF or alcoholic formalin.
Oxidation, fading, and/or disappearance of the SISH signal may be due to certain brands of mounting media. See Table 4 for information regarding compatibility of mounting
media.
5.
The PTEN DNP probe was developed to stain tissue sections that are cut at ~4 μm in thickness.2
TROUBLESHOOTING
Issue
Solution
Positive control is negative.
Check that the slide has the proper barcode label.
Positive control is negative or exhibits weaker staining than
expected.
Check other positive controls stained on the same staining run to determine if the failure is due to the control slide
or reagents used. Every specimen contains internal positive controls, and thus the presence of appropriate SISH
staining in normal cells indicates positive staining. Positive control specimens are recommended for potential
troubleshooting for each run.
Specimen staining is weak or absent.
Specimens may have been improperly collected, fixed, or stored. See the Specimen Preparation section.
· If the sample is fixed as recommended but staining is weak, use ISH Protease 3 for more than 20 minutes or ISH
Protease 2 for 4 minutes or more.
· Increase Silver C time. Note that background staining may increase as Silver C incubation time increases.
· Check the reagent dispenser priming chamber or meniscus for foreign materials or particulates, such as fibers or
precipitates. If the dispenser is blocked, do not use the dispenser and contact your local support representative.
Otherwise, re-prime the dispenser by aiming the dispenser over a waste container, removing the nozzle cap, and
pressing down on the top of the dispenser.
· Increase concentration of probe used.
Background staining interferes with enumeration of
samples.
Use ISH Protease 3 for 12 minutes instead of 20 minutes.
Decrease Silver C time to 4 minutes
Decrease probe concentration in dispenser
Add additional blocking DNA to dispenser
Sections thicker than 4 μm exhibit nuclear bubbling due to
excess paraffin.
Select the “Extended” deparaffinization option in the staining procedure.
Table 4. Compatibility of Mounting Media with SISH-based assays.
Mounting Media
Manufacturer
Type (Xylene, alcohol, aqueous)
Compatibility with SISH
Entellan
Merck
Xylene
No
Entellan New
Merck
Xylene
No
Eukitt
EMS
Xylene
No
HSR
Sysmex
Xylene
No
Malinol
Muto Chemical
Xylene
No
Acrytol
SurgiPath
Xylene
Yes
Alcolmount
Diapath
Alcohol
Yes
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Mounting Media
Manufacturer
Type (Xylene, alcohol, aqueous)
Compatibility with SISH
BioMount 2
BBInternational
Xylene
Yes
Cytoseal 60
Richard Allan Scientific
Xylene
Yes
Diamount
Diapath
Xylene
Yes
DPX
BDH: Raymond Lamb
Xylene
Yes
FloTexx
Lerner Labs
Xylene
Yes
Gel Mount
Biomeda
Aqueous
Yes
Histomount
Raymond Lamb
Xylene
Yes
MicroMount
SurgiPath
Xylene
Yes
MM24
SurgiPath
Xylene
Yes
Mountex
Histolab
MountQuick
Daido Sangyo Co.
Paramount
Protaqs Quartett: Dako
Permount
Fisher
Xylene
Yes
Pertex
Cell Path
Xylene
Yes
Shandon Consul mount
Thermo Scientific
Softmount
WAKO
SureMount
Thermo EZ Mount
Ultramount
Xylene
Yes
Aqueous
Yes
Xylene
Yes
Xylene
Yes
Lemasol A
Yes
Triangle Biomedical Sciences
Xylene
Yes
Thermo Scientific
Xylene
Yes
Dako
Xylene
Yes
PERFORMANCE CHARACTERISTICS
The performance of the PTEN DNP Probe was evaluated through specificity studies. All staining was performed using the ULTRA Dual Color Open Probe protocol as noted in Table 2
on BenchMark ULTRA instruments unless otherwise specified. Data on file.
REFERENCES
1.
2.
Carson F, Hladik C. Histotechnology: A Self Instructional Text, 3rd edition. Hong Kong: American Society for Clinical Pathology Press; 2009.
Roche PC, Hsi ED. Immunohistochemistry-Principles and Advances. Manual of Clinical Laboratory Immunology, 6th edition. In: NR Rose, ed. ASM Press; 2002.
INTELLECTUAL PROPERTY
VENTANA, BENCHMARK, ultraView, and the VENTANA logo are trademarks of Roche.
All other trademarks are the property of their respective owners.
Ventana Medical Systems, Inc. grants to the Purchaser a single use only license under U.S. Pat. Nos. 6045759, 6945128, and 7378058, and any foreign counterparts.
© 2012 Ventana Medical Systems, Inc.
CONTACT INFORMATION
Contact your local support representative.
Ventana Medical Systems, Inc.
1910 E. Innovation Park Drive
Tucson, Arizona 85755
USA
+1 520 887 2155
+1 800 227 2155 (USA)
www.ventana.com
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