ChromoMap鈩? Blue Kit
Biotin-free NBT/BCIP Detection Kit for the Ventana DISCOVERY庐 Series of Instruments
Catalog Number: 760-161
125 Tests
Intended Use
For Research Use Only. Not for in vitro Diagnostic Use.
Introduction
Automated immunohistochemistry (IHC) is a powerful tool for demonstrating the presence of unique antigens in tissues and cells.
The ChromoMap鈩? Blue Kit was designed for biotin-free antigen localization in frozen sections or formaldehyde-fixed, paraffin-
庐
embedded tissue sections, to be used in conjunction with the DISCOVERY series of instruments and Ventana Medical Systems鈥?
ancillary reagents for optimal performance.
Kit Components
Test
Catalog # Description Components (Part #) Storage
Size
鈥? Activator CM 125
(For increasing signal intensity)
鈥? 2-8掳 C
NBT CM
760-161 ChromoMap Blue kit 125
(Do not freeze)
(Hue enhancer)
鈥? BCIP CM 125
(Substrate for Alkaline Phosphatase)
An appropriate Conjugates and the ChromoMap Blue Kits are both required to obtain IHC results. The components can be ordered
together or individually. Bellow is a list of the related components that need to be run with the ChromoMap Blue Kit to obtain IHC
results.
Test
Description Components (Catalog #) Storage
Size
2-8掳 C
鈥? 125
UltraMap鈩? anti-Rb Alk Phos (760-4314)
UltraMap Alk Phos (Do not freeze)
conjugates 2-8掳 C
鈥? 125
UltraMap鈩? anti-Ms Alk Phos (760-4312)
(Do not freeze)
Instructions for Use
Register the kit on your Discovery series instrument, as described in the on-screen directions. Open the package, and take out the
dispensers. Remove the cap from the nozzle of each dispenser, and place it on the nozzle cap holder on the rear of the dispenser.
While holding the dispenser upright, remove the yellow shipping key by pulling the key tab to disengage it from each end. Do not
cover the nozzle tip or depress the dispenser while removing key. Place the dispensers on the reagent tray, along with the
appropriate accessory reagents.
The DISCOVERY series of instruments and both the UltraMap Alk Phos Conjugates and the ChromoMap Blue Kit form an
integrated system. ALL KIT COMPONENTS MUST BE USED TOGETHER in order to obtain high-quality and consistent results.
Omitting or changing any of the solutions may compromise the final outcome.
Bulk reagents should be prepared using quality reagent-grade water, not tap water. Carboys for storing bulk reagents should be
rinsed thoroughly between fillings.
June 2006 1
Ventana Translational Diagnostics Group
http://www.ventanadiscovery.com
Controls
Staining results can be affected by endogenous phosphatase activity. Therefore, it is important to include a negative control on
every tissue tested to identify areas of endogenous phosphatase activity and/or nonspecific binding of antibody. When this is done,
the specificity of the staining reaction can be documented by comparing the negative control staining to the primary antibody
staining. In addition, a known positive tissue should be run with every assay. The staining of the positive control serves as a
baseline for evaluating run-to-run and/or day-to-day consistency.
Protocols
Although general tissue processing protocols are similar among laboratories, a single universal protocol is not in place, thus no two
laboratories prepare tissue samples in exactly the same way. Tissue processing has the greatest single impact on the end result,
and different tissue types often require slightly different pretreatments for optimum results. It is not surprising then that there is not a
single protocol that is optimal for all cases.
The Closed Loop Assay Development (CLAD) process for IHC empowers the user to optimize development protocols based on
crisp morphology, signal intensity and high signal to noise ratio. This allows for consistent and reproducible results for both routine
and complex projects, and can serve as a guideline for optimizing UltraMap anti-Rb Alk Phos and UltraMap anti-Ms Alk Phos
protocols. The CLAD flowchart can be found in the package insert for the UltraMap Alk Phos conjugates.
NexES庐 Protocol Editor Window
1. Open the NexES software.
2. To create a protocol, click on the 鈥淧rotocols鈥? button on the main screen. A window will appear on the screen with 鈥淐reate/Edit
Protocol鈥? and 鈥淢anage Protocol鈥?. Click on 鈥淐reate/Edit Protocol鈥? to open the 鈥淣exES Protocol Editor鈥? window.
3. Select the 鈥淩esearch IHC UltraMap AP鈥? procedure under the 鈥淧rocedure鈥? field.
Saving the Protocol
1. After all options have been selected (i.e., no more yellow boxes), click on the 鈥淪ave As鈥? button. Fields will appear for a protocol
name and protocol number. Type in an unused name for the protocol, and select an unused number in the appropriate boxes.
Click on the 鈥淪ave鈥? button, and the protocol will be saved.
Preparing Labels and Loading Slides
1. From the toolbar on the bottom of the main screen, click on the bar code symbol. Click on the 鈥淧rotocols鈥? button. Highlight the
protocol number and name desired in the protocol field. Click on the 鈥淎dd>>鈥? button once for each protocol bar code label you
want to print. Click on the 鈥淐lose/Print鈥? button. Enter any additional information you want to appear on the label in the "User
Prompt" fields. Click the 鈥淧rint鈥? button. When the last bar code has been printed, click on the 鈥淓xit鈥? button.
2. Place the bar code(s) on the slide(s), load them carefully onto the instrument, close the instrument, and click on the 鈥淩un鈥?
button. Click on the 鈥淩eagents/Reagent Tray Loaded鈥? box and 鈥淩eagent Caps Removed鈥? box. Enter the number of slides
loaded, and click on 鈥淪tart Run鈥?.
End of Run Instructions
1. All runs will go into a hold step before Counterstain/Slide Cleaning. To complete the run, press the logo button on the
instrument. If Counterstain and/or Slide Cleaning were selected in the protocol, the instrument will perform these functions
then stop. If Counterstain and/or Slide Cleaning were not selected, the instrument will 鈥渉ome,鈥? and then the run will end.
2. When the run ends, open the instrument and collect each slide in a slide holder previously filled with Reaction Buffer. Rinse off
the slides by immersing them in a solution made of a few drops of dish soap and warm water. Rinse the slides thoroughly
before dehydration of the tissue through a battery of increasing concentration of Alcohol and Xylene. Coverslip the slides in
mounting media with glass coverslips. Air-dry before use.
June 2006 2
Ventana Translational Diagnostics Group
http://www.ventanadiscovery.com
IHC Troubleshooting
Problem Possible Cause Next Step
1. Insufficient cell conditioning 1. Increase cell conditioning time
No Signal
2. Inadequate protease digestion 2. Use stronger protease
Good Tissue Morphology
OR
Extend protease digestion time
1. Over cell conditioning 1. Decrease or remove cell conditioning steps
No Signal
Poor Tissue Morphology
2. Over digested 2. Use weaker protease or shorter digestion time
1. Insufficient cell conditioning 1. Increase cell conditioning time
2. Inadequate protease digestion 2. Use stronger protease
OR
Weak Signal
Extend protease digestion time
Low Background
3. Antibody too dilute 3. Increase antibody concentration
OR
Extend antibody incubation time
1. Non-specific binding of the primary 1. Use blocking reagent during the primary
antibody incubation step
Weak Signal
2. Antibody concentration too high
High Background
2. Decrease the antibody concentration and
increase the incubation time
1. Antibody concentration too high 1. Decrease antibody concentration
Signal Too Strong
AND/OR
Low Background
Decrease antibody incubation time
1. Antibody concentration too high 1. Decrease antibody concentration
Signal Too Strong and/or decrease antibody incubation time
High Background
2. Over cell conditioning 2. Decrease or remove cell conditioning steps
Intellectual Property
庐 庐 庐
UltraMap鈩? and ChromoMap鈩? are trademarks of Ventana Medical Systems, Inc. DISCOVERY , NexES , and VENTANA are
registered trademarks of Ventana Medical Systems, Inc.
Ventana grants to Purchaser a single-use only license under the following patents:
U.S. Pat. Nos. 6045 759, 6192 945 B1, 6416 713 B1 and foreign counterparts.
UltraMap anti-Rb Alk Phos and UltraMap anti-Ms Alk Phos are covered by patents pending.
Technical Consultation
Additional technical information can be obtained from our Molecular Discovery Systems Technical Center at:
U.S.A. : Europe :
(800) 227-2155 (+33) 3 90 40 52 00
Ventana Medical Systems, Inc. Ventana Medical Systems S.A.
1910 E. Innovation Park Drive Parc d鈥橧nnovation
Tucson, AZ 85755 Rue G. de Kaysersberg
USA BP 30144
F-67404 ILLKIRCH Cedex
France
Japan : Australia :
(+81) 45 228 5071 +61 (0) 3 9431 6064
Ventana Japan K.K. Ventana Medical Systems, Pty. Ltd.
Landmark Tower, 35F 5/39 Grand Boulevard
2-2-1, Minato Mirai, Nishi-ku Montmorency, Victoria 3094
Yokohama, Kanagawa 220-8135 Australia
Japan
June 2006 3
Ventana Translational Diagnostics Group
http://www.ventanadiscovery.com
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