30. 20 L carboy
31. 1 or 2 L graduated cylinder
* As needed for specific applications.
10X SSC
Storage and Handling
Catalog number 950-110 Store at room temperature (20掳 to 28掳C) out of direct sunlight. Do not freeze. The user
must validate any storage conditions other than those specified in the package insert.
This reagent is expiration dated. When properly stored, the reagent is stable to the date
INDICATIONS AND USE
indicated on the label. Do not use reagent beyond the expiration date for the prescribed
Intended Use
storage method.
This reagent is intended for in vitro diagnostic use.
There are no definitive signs to indicate instability of this product; therefore, positive and
Ventana Medical Systems' (Ventana庐) 10X SSC, Sodium Chloride Sodium Citrate buffer
negative controls should be run simultaneously with unknown specimens. Your local
solution is used for stringency washes and to rinse slides between staining steps and
Ventana office should be contacted immediately if there is an indication of reagent
provide a stable aqueous environment for the in situ hybridization reactions carried out on a
instability.
Ventana automated slide stainer.
Specimen Collection and Preparation for Analysis
Summary and Explanation
For tissue specimens, routinely processed, formalin fixed, paraffin embedded tissues are
10X SSC must be diluted prior to use.
suitable for use with this reagent when used with Ventana detection kits and a Ventana
Once diluted, 2X SSC buffer solution is poured into the appropriate position (SSC) of the
automated slide stainer (see Materials and Reagents Needed, But Not Provided section).
automated fluidics module on the Ventana automated slide stainer. The instrument applies
The recommended tissue fixative is 10% neutral buffered formalin. Variable results may
2X SSC automatically as required by the procedure being run. For additional information
occur as a result of prolonged fixation or special processes such as decalcification of bone
refer to the automated slide stainer Operator鈥檚 Manual.
marrow preparations.
Each section should be cut the appropriate thickness and placed on a positively charged
Principles and Procedures
glass slide. Slides containing the tissue section may be baked for at least 2 hours (but not
The reliability of nucleic acid binding is controlled by stringency of the hybridization
longer than 24 hours) in a 70掳 C 卤 5掳 C oven.
environment. One of the major controlling factors of stringency is the salt concentration.
Salt provides ions that partially mask the negative charge on the phosphate backbone of
For cytology specimens, use either TriPath Imaging, Inc.s鈥? AutoCyte Prep System or Cytyc
DNA and RNA. The higher the salt concentration the lower the stringency which translates
Corporations鈥? ThinPrep Pap Test specimen collection systems which are collection systems
to less energy required to hold two strands of nucleic acid together. 10X SSC is a sodium
specifically designed for certain cytology specimens. Cytology specimens must be collected
citrate, sodium chloride buffer that needs to be used at a final 2X working concentration for
and placed in the appropriate preservative and slide preparation must be performed
hybridization reactions. The Ventana automated slide stainer automatically further dilutes
according to the manufactures鈥? instructions.
the 2X SSC solution as needed for stringency washes.
WARNINGS AND PRECAUTIONS
Technical Note: 10X SSC comes as a 5X concentrate (10X salt concentrate) which when
1. Take reasonable precautions when handling reagents. Use disposable gloves when
diluted five fold provides a 2X SSC buffer for hybridization reactions.
handling suspected carcinogens or toxic materials.
2. Avoid contact of reagents with eyes and mucous membranes. If reagents come in
MATERIALS AND METHODS
contact with sensitive areas, wash with copious amounts of water.
Reagents Provided
3. Do not smoke, eat or drink in areas where specimens or reagents are being handled.
One 2 liter bottle of 10X SSC contains sodium chloride, sodium citrate and a preservative.
4. Patient specimens and all materials contacting them should be handled as
biohazardous materials and disposed of with proper precautions. Never pipette by
Reconstitution, Mixing, Dilution, Titration
mouth.
10X SSC must be diluted with four parts of distilled or deionized water prior to use.
5. Avoid microbial contamination of reagents, as this could produce incorrect results.
1. Pour the entire contents of the 2 liter10X SSC solution into the 20 liter carboy.
6. This reagent has been optimally formulated for a 1:5 (2X) dilution. Further dilution
2. Using a 1 or 2 liter graduated cylinder, add 8 liters of deionized or distilled water to
may result in poor instrument performance and loss of staining. Any such change
the 20 liter carboy. One bottle of 10X SSC provides a final volume of 10 liters.
must be validated by the user.
3. If large quantities of bubbles form during the filling procedure, allow the solution to
7. When used according to instructions, this product is not classified as a hazardous
sit until the bubbles have dissipated.
substance. The preservative in the reagent is ProClin 300, containing the active
4. Place the cap on the container, mix the solution thoroughly for 30 minutes. The
ingredients 5-chloro-2-methyl-4-isothiazine-3-one and 2-methyl-4-isothiazolin-3-one.
diluted 2X SSC solution is ready to use on the automated stainer.
Symptoms of overexposure to ProClin 300 include skin and eye irritation, and
irritation of mucous membranes and upper respiratory tract. The concentration of
Materials and Reagents Needed But Not Provided
ProClin 300 in this product is 0.05% and does not meet the OSHA criteria for a
The following reagents and materials may be required but are not provided with this kit:
hazardous substance. Systemic allergic reactions are possible in sensitive
5. Positive and negative tissue controls
individuals.
6. RNA Positive Control or ISH DNA Negative Control Probe
8. This reagent may be harmful if swallowed or inhaled. This reagent may cause short
AutoCyte庐 Prep System specimen collection system, TriPath Imaging, Inc.
1.
term skin or eye irritation and may be irritating to mucous membranes and the
ThinPrep庐 Pap TestTM specimen collection system, Cytyc Corporation
7. respiratory tract. Inhalation of high vapor or aerosol concentrations may result in
8. Microscope slides, positively charged headaches, dizziness, anesthesia, drowsiness, unconsciousness and other central
9. Drying oven capable of maintaining a temperature of 70掳 C 卤 5掳 C
nervous system symptoms. OSHA鈥檚 permissible exposure limit is 5 mg/m3 (oil mist).
10. Bar code labels (appropriate for negative control and primary antibody being tested)
9. 10X SSC is not flammable.
11. 10% neutral buffered formalin
12. Staining jars or baths
INSTRUCTIONS FOR USE
13. Timer
The 2X SSC solution is poured into the appropriate position (SSC) of the automated fluidics
14. Xylene
module on the Ventana automated slide stainer. The 2X SSC solution is applied
15. Ethanol or reagent alcohol
automatically as required by the procedure being run to rinse slides between staining steps
16. Deionized or distilled water
and maintain a stable aqueous environment on the slide.
BenchMark庐 and BenchMark庐 XT automated slide stainers
17.
ISH iVIEWBlueTM solution
18. Prior to initial use of the 2X SSC solution in the user鈥檚 laboratory, appropriate staining
19. ISH Signal Clarifier* should be verified by staining a number of positive and negative controls. Ventana
20. Probe recommends positive controls be placed on the same slide as the patient sample sample.
EZ PrepTM* Variable results may occur due to sample fixation. The clinical interpretation of any staining
21.
or its absence must be complemented by morphological studies and evaluation of proper
22. LCS, coverslip solution
controls. Evaluation must be made by a qualified pathologist within the context of the
23. Reaction Buffer
patient鈥檚 clinical history and other diagnostic tests.
24. Cell Conditioning 1 (CC1) or Cell Conditioning 2 (CC2)*
25. ISH Protease 1, 2 or 3*
26. ISH Red Counterstain Step by Step Procedure
Ventana reagents have been developed for use on a Ventana automated slide staining
27. Mounting medium
system in combination with Ventana detection kits and accessories. Recommended
28. Cover glass
staining protocols for the automated slide stainers are described in the package insert of
29. Light microscope (20-80X)
2003-11-30 Page 1 of 3 14827EN Rev A
the probe of interest. The parameters for the automated procedures can be displayed,
printed and edited according to the procedure in the Operator鈥檚 Manual. Other operating Interpretation of Results
parameters for the automated slide stainers have been preset at the factory. For more The Ventana automated staining procedures cause colored reaction products. Refer to the
detailed instructions and additional protocol options refer to your Operator鈥檚 Manual. appropriate detection kit package insert for expected color reactions. A qualified pathologist
experienced must evaluate positive and negative controls before interpreting results.
BenchMark or BenchMark XT Automated Slide Stainers
1. Apply slide bar code label which corresponds to the probe protocol to be performed. Positive Tissue Control
2. Load the probe, appropriate detection kit and required accessory reagents onto the The stained positive sample control should be examined first to ascertain that all reagents
reagent tray and place them on the automated slide stainer. Check bulk fluids and are functioning properly. The presence of an appropriately colored reaction product within
waste. the target cells is indicative of positive reactivity. Refer to the package insert of the
3. Load the slides onto the automated slide stainer. detection kit used for expected color reactions. Intensity of the counterstain will be
4. Start the staining run. depended on the incubation time selected.
5. At the completion of the run, remove the slides from the automated slide stainer. For in situ hybridization counterstaining the incubation length and potency of the ISH Red
6. For ISH iVIEWBlue detection kit, wash in a mild dishwashing detergent or alcohol to used will range from a pale to dark pink coloration of cell nuclei.
Excessive or incomplete counterstaining may compromise proper interpretation of results.
remove the coverslip solution.
If the positive tissue control fails to demonstrate positive staining, any results with the test
7. Transfer the slides into a bath of distilled water for approximately 1 to 3 minutes.
specimens should be considered invalid.
Shake off excess water.
8. View the level of this and all subsequent baths. Make sure that the solutions will
Patient Tissue
completely cover the slides in the rack. Add new reagent to each container in
Patient specimens should be examined last. Positive staining intensity should be assessed
sufficient quantity to cover the slides at all times. Be sure to remove excess fluid after
within the context of any background staining. As with any probe test, a negative result
each step.
means that the RNA or DNA sequence targeted was either not detected or the copy
9. Transfer the slides to a 90% ethanol for approximately 1 to 3 minutes.
number was below the sensitivity level of the kit, not that the sequence is absent in the cells
10. Transfer the slides to a 100% ethanol for approximately 1 to 3 minutes.
assayed. The morphology of each sample should also be examined utilizing a hematoxylin
11. Transfer the slides into a second bath of 100% ethanol for approximately 1 to 3
and eosin stained section when interpreting any immunohistochemical, in situ or special
minutes.
stains result. The patient's morphologic findings and pertinent clinical data must be
12. Transfer the slides into the first xylene bath for approximately 1 to 3 minutes.
interpreted by a qualified pathologist.
13. Transfer the slides into a second xylene bath for approximately 1 to 3 minutes. The
slides may be left in this xylene bath until they are cover slipped.
14. Return all covers to dishes and turn off the fume hood. LIMITATIONS
General Limitations
1. ISH is a multiple step diagnostic process that requires specialized training in the
Quality Control Procedures
selection of the appropriate reagents, samples, fixation, processing, preparation of
Positive Tissue Control
the slide, and interpretation of the staining results.
A positive sample control must be run with every staining procedure performed.
2. Sample staining is dependent on the handling and processing of the sample prior to
The positive staining sample components are used to confirm that the reagents were
staining. Improper fixation, freezing, thawing, washing, drying, heating, sectioning, or
applied and the instrument functioned properly. This sample may contain both positive and
contamination with other samples or fluids may produce artifacts or false negative
negative staining cells or tissue components and serve as both the positive and negative
results. Inconsistent results may result from variations in fixation and embedding
control. Control samples should be fresh autopsy, biopsy or surgical specimens prepared or
methods, or from inherent irregularities within the sample.
fixed as soon as possible in a manner identical to the test sections. Such samples may
3. Excessive or incomplete counterstaining may compromise proper interpretation of
monitor all steps of the procedure, from sample preparation through staining. Use of a
results.
tissue section fixed or processed differently from the test specimen will provide control for
4. The clinical interpretation of any positive staining, or its absence, must be evaluated
all reagents and method steps except fixation and tissue processing.
within the context of clinical history, morphology and other histopathological criteria.
A sample with weak positive staining is more suitable for optimal quality control and for
The clinical interpretation of any staining, or its absence, must be complemented by
detecting minor levels of reagent degradation.
morphological studies and proper controls as well as other diagnostic tests. It is the
Known positive sample controls should be utilized only for monitoring the correct
responsibility of a qualified pathologist to be familiar with the reagents and methods
performance of processed sample and test reagents, not as an aid in determining a specific
used to produce the stained preparation. Staining must be performed in a certified
diagnosis of patient samples. If the positive sample controls fail to demonstrate positive
licensed laboratory under the supervision of a pathologist who is responsible for
staining, results with the test specimens should be considered invalid.
reviewing the stained slides and assuring the adequacy of positive and negative
controls.
Negative Tissue Control
The same sample used for the positive sample control may be used as the negative sample 5. Ventana provides reagents at optimal dilution for use when the provided instructions
are followed. Any deviation from recommended test procedures may invalidate
control. The variety of cell types present in most sample sections offers internal negative
expected results. Appropriate controls must be employed and documented. Users
control sites, but this should be verified by the user. The components that do not stain
who deviate from recommended test procedures must accept responsibility for
should demonstrate the absence of specific staining, and provide an indication of non
interpretation of patient results.
specific background staining. If specific staining occurs in the negative sample control sites,
6. Reagents may demonstrate unexpected reactions in previously untested samples.
results with the patient specimens should be considered invalid.
The possibility of unexpected reactions even in tested sample groups cannot be
completely eliminated because of biological variability of antigen expression in
Unexplained Discrepancies
neoplasms, or other pathological samples. Contact your local Ventana office with
Unexplained discrepancies in controls should be referred to your local Ventana office
documented unexpected reactions.
immediately. If quality control results do not meet specifications, patient results are invalid.
See the Troubleshooting section of this insert. Identify and correct the problem, then repeat
Specific Limitations
the patient samples.
1. This reagent must be examined for microbial contamination prior to use. The signs
indicating contamination or instability of this product are: turbidity of the solution,
Negative Reagent Control
odor development or precipitation. At the first sign of possible reagent contamination
For in situ hybridization a negative control must be run. Ventana recommends following the
or instability, call your local Ventana Office.
quality control recommendations of the College of American Pathologists Laboratory
2. This reagent has been optimally formulated for a 1:5 (2X) dilution. Further dilution
Accreditation Program, Anatomic Pathology Checklist.
may result in poor instrument performance and loss of staining. Any such change
must be validated by the user.
Assay Verification
Prior to initial use of a staining system in a diagnostic procedure, the specificity of the
SUMMARY OF EXPECTED RESULTS
system should be verified by testing it on a series of samples with known staining
performance characteristics representing known positive and negative samples (refer to the Refer to the appropriate Ventana probe package insert for expected patient sample results.
Appropriate sample control results verify the reagents and system are working properly.
Quality Control Procedures previously outlined in this section of the product insert, to the
Quality Control recommendations of the College of American Pathologists Laboratory
Accreditation Program, Anatomic Pathology Checklist, and the NCCLS Approved TROUBLESHOOTING
Guideline). These quality control procedures should be repeated for each new probe lot, or 1. If the positive control exhibits weaker staining than expected, other positive controls
run during the same instrument run should be checked to determine if it is because of
whenever there is a change in assay parameters.
the probe or one of the common secondary reagents.
2003-11-30 Page 2 of 3 14827EN Rev A
2. If the positive control is negative, it should be checked to ensure that the slide has the CONTACT INFORMATION
proper bar code label. If the slide is labeled properly, other positive controls run on North America
the same instrument run should be checked to determine if it is because the 10X Ventana Medical Systems, Inc.
SSC was too dilute, other reagents in the automated fluidics module may have been 1910 Innovation Park Drive
transposed or samples may have been improperly collected, fixed or deparaffinized. Tucson, Arizona 85737
3. If excessive background staining occurs, high levels of endogenous biotin may be U.S.A.
present. Call your local Ventana office. 800 227 2155 (U.S. only)
4. If all of the paraffin has not been removed, the deparaffinization procedure should be 520 229 1910
repeated.
5. If specific probe staining is too intense, the run should be repeated with incubation Europe
time shortened by intervals of 4 minutes until the desired stain intensity is achieved. Ventana Medical Systems, S.A.
6. If sample sections wash off the slide, slides should be checked to ensure that they Parc d鈥橧nnovation 鈥? BP 30 144
are positively charged. Rue G. de Kaysersberg
7. For corrective action, refer to the Step By Step Procedure section, the automated F - 67404 Illkirch Cedex
slide stainer Operator鈥檚 Manual or contact your local Ventana office. France
33 (0) 3 90 40 52 00
REFERENCES
Beesely JE. Immunocytochemistry and In Situ Hybridization in the Biomedical Sciences. EU Representative
Birkh盲user, 2001. MDCI Ltd.
College of American Pathologists Laboratory Accreditation Program, Anatomic Pathology Arundel House
Checklist, 2001. 1 Liverpool Gardens
NCCLS. Quality Assurance for Immunocytochemistry: Approved Guideline. NCCLS Worthing
document MM4-A- (ISBN 1-56238-396-5). NCCLS, 940 West Valley Road, Suite 1400, West Sussex BN11 1SL
Wayne, PA 19087-1898 USA, 1999. UK
Herman GE, Elfont EA. The taming of immunohistochemistry: the new era of quality control.
Biotech Histochem 66(4): 194-199, 1991. Japan
Roche PC, Hsi ED. Immunohistochemistry-Principles and Advances. Manual of Clinical Ventana Japan K.K.
Laboratory Immunology, 6th edition. (NR Rose Ed.) ASM Press, 2002. Landmark Tower, 35F
2-2-1, Minatomirai, Nishi-ku
Yokohama, 220-8135
INTELLECTUAL PROPERTY
Japan
iVIEWTMand EZ PrepTM and are trademarks of Ventana Medical Systems, Inc.;
81 (0) 45-228-5071
BenchMark庐 and Ventana庐 are registered trademarks of Ventana Medical Systems, Inc.
Australia, New Zealand
Covered by the following patents: U.S. Pat. Nos. 6045 759, 6192 945 B1, 6416 713 B1 and
Ventana Medical Systems, Pty Ltd
foreign counterparts.
5/39 Grand Boulevard
Montmorency VIC 3094
AutoCyte庐 is a registered trademark of TriPath Imaging, Inc.; ThinPrep庐 is a registered
Australia
trademark of Cytyc Corporation and ThinPrep庐 Pap TestTM is a trademark of Cytyc 61 (0) 3 9431 6064
Corporation.
2003-11-30 Page 3 of 3 14827EN Rev A
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