OmniMap鈩? anti-Rb HRP
OmniMap鈩? anti-Ms HRP
Biotin-free DAB Detection Systems for the Ventana DISCOVERY庐 Series of Instruments
Catalog Number: 760-4311 (OmniMap anti-Rb HRP)
760-4310 (OmniMap anti-Ms HRP)
125 Tests
Intended Use
For Research Use Only. Not for in vitro Diagnostic Use.
Introduction
Since its invention in the 1940s, immunohistochemistry (IHC) has become a routine and essential tool for many laboratories across
th
many applications. Automation of IHC in the late 20 century increased the power of this tool for demonstrating the presence of
unique antigens in tissue and cells.
One of the most routine IHC methods is the use of streptavidin conjugates to target the antigen of interest. Streptavidin-horseradish
peroxidase conjugate is used to catalyze the 3,3鈥?-Diaminobenzidine tetrahydrochloride (DAB) / H2O2 reaction to produce an
insoluble dark brown precipitate that can be visualized.
OmniMap HRP anti-Rb and OmniMap HRP anti-Ms are biotin-free conjugates based on proprietary multimer technology. They
consist of a robust chemistry that provides clean background in combination with enhanced specificity and sensitivity, which
increases the signal-to-noise ratio. They are designed to be used in conjunction with the DISCOVERY series of instruments and
Ventana Medical Systems鈥? ancillary reagents for optimal performance.
Kit Components
For flexibility, the detection systems are split between the specific conjugate, OmniMap HRP conjugate and the universal DAB
chromogenic kit, ChromoMap DAB. Therefore, the same chromogenic kit can be used with two or more conjugates on the same
instrument run. Conjugate and ChromoMap DAB can be ordered together or individually; however, both are required to obtain IHC
results. Refer to the Kit Components table for further information.
Description Components (Catalog #) Test Storage
Size
鈥? 125
OmniMap anti-Rb HRP (760-4311) 2-8掳 C
OmniMap HRP conjugate
(Do not freeze)
鈥? 125
OmniMap anti-Ms HRP (760-4310)
Related Components
Both the Conjugate and the ChromoMap DAB are 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 specific conjugates to obtain IHC results.
Catalog # Description Components (Part #) Test Storage
Size
鈥? Inhibitor CM 125
(For reducing the endogenous peroxidase activity)
鈥? DAB CM 125
2-8掳 C
(Diaminobenzidine in stabilizer solution with preservative)
760-159 ChromoMap DAB kit
鈥? (Do not freeze)
DAB H2O2 CM 125
(Substrate for peroxidase)
鈥? Copper CM 125
(Copper sulfate solution)
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Instructions for Use
Register the kits 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 OmniMap HRP Conjugate and the ChromoMap DAB Kits 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.
Controls
DAB staining results can be affected by endogenous peroxidase activity. Therefore, it is important to include a negative control on
every tissue tested to identify areas of endogenous peroxidase 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 (see below) 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 OmniMap anti-Rb HRP and Omni anti-Ms HRP protocols.
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NexES庐 Protocol Editor Window
Note: The OmniMap anti-Rb HRP and OmniMap anti-Ms HRP detection systems can only be run with NexES
software version 9.0 or higher. Please contact Technical Customer Care if a software upgrade is required.
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es IHC Omni-UltraMap HRP鈥? or 鈥淩es IHC Omni-UltraMap HRP XT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 and
air-dry before use.
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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
OmniMap鈩? is a trademark 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.
OmniMap anti-Rb HRP and OmniMap anti-Ms HRP are covered by patents pending.
Technical Consultation
Additional technical information can be obtained from Technical Customer Care at:
(800) 227-2155 (+33) 3 90 40 52 00
U.S.A. : Europe :
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
(+81) 45 228 5071 +61 (0) 3 9431 6064
Japan : Australia :
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
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