UltraMap Alk Phos. UltraMap anti-ms Alk Phos

Transcription

1 UltraMap anti-rb Alk Phos UltraMap anti-ms Alk Phos Biotin-free Alkaline Phosphatase Detection Systems for the Ventana DISCOVERY Series of Instruments Catalog Number: s (UltraMap anti-rb Alk Phos) (UltraMap anti-ms Alk Phos) 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 many applications. Automation of IHC in the late 20 th century increased the power of this tool for demonstrating the presence of unique antigens in tissue and cells. UltraMap anti-rb Alk Phos and UltraMap anti-ms Alk Phos are biotin-free detection systems 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 ancillary reagents for optimal performance. Kit Components For flexibility, the detection systems are split between the specific conjugate, UltraMap Alk Phos conjugates and the universal chromogenic kits, ChromoMap Blue and ChromoMap Red. Therefore, the same chromogenic kit can be used with two or more conjugates on the same instrument run and likewise the same UltraMap Alk Phos conjugate can be used with ChromoMap Blue and ChromoMap Red on the same instrument. Conjugates and ChromoMap kits can be ordered together or individually; however, both are required to obtain IHC results. Refer to the following tables for further information. Description Components (Catalog #) UltraMap Alk Phos conjugates UltraMap anti-rb Alk Phos ( ) UltraMap anti-ms Alk Phos ( ) Ventana Translational Diagnostics Group June

2 Related Components Both the Conjugate and either ChromoMap Blue or ChromoMap Red 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 #) ChromoMap Blue kit Activator CM (For increasing signal intensity) NBT CM (Hue enhancer) BCIP CM (Substrate for Alkaline Phosphatase) Catalog # Description Components (Part #) ChromoMap Red kit Instructions for Use Activator R CM (For increasing signal intensity) Fast Red CM (Hue enhancer) Naphthol CM (Substrate for Alkaline Phosphatase) 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 conjugate and the ChomoMap 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. 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 (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 UltraMap anti-rb Alk Phos and UltraMap anti-ms Alk Phos protocols. Ventana Translational Diagnostics Group June

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4 NexES Protocol Editor Window Note: The UltraMap anti-rb Alk Phos and UltraMap anti-ms Alk Phos 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 Protocols button on the main screen. A window will appear on the screen with Create/Edit Protocol and Manage Protocol. Click on Create/Edit Protocol to open the NexES Protocol Editor window. 3. Select the Research IHC UltraMap AP procedure under the Procedure field. Saving the Protocol 1. After all options have been selected (i.e., no more yellow boxes), click on the Save As 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 Save 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 Protocols button. Highlight the protocol number and name desired in the protocol field. Click on the Add>> button once for each protocol bar code label you want to print. Click on the Close/Print button. Enter any additional information you want to appear on the label in the "User Prompt" fields. Click the Print button. When the last bar code has been printed, click on the Exit button. 2. Place the bar code(s) on the slide(s), load them carefully onto the instrument, close the instrument, and click on the Run button. Click on the Reagents/Reagent Tray Loaded box and Reagent Caps Removed box. Enter the number of slides loaded, and click on Start Run. 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 home, 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. Ventana Translational Diagnostics Group June

5 IHC Troubleshooting Problem Possible Cause Next Step No Signal Good Tissue Morphology No Signal Poor Tissue Morphology 1. Insufficient cell conditioning 2. Inadequate protease digestion 1. Over cell conditioning 2. Over digested 1. Insufficient cell conditioning 1. Increase cell conditioning time 2. Use stronger protease Extend protease digestion time 1. Decrease or remove cell conditioning steps 2. Use weaker protease or shorter digestion time 1. Increase cell conditioning time Weak Signal Low Background Weak Signal High Background Signal Too Strong Low Background Signal Too Strong High Background 2. Inadequate protease digestion 3. Antibody too dilute 1. Non-specific binding of the primary 2. Antibody concentration too high 2. Use stronger protease Extend protease digestion time 3. Increase antibody concentration Extend antibody incubation time 1. Use blocking reagent during the primary antibody incubation step 2. Decrease the antibody concentration and increase the incubation time 1. Antibody concentration too high 1. Decrease antibody concentration AND/ Decrease antibody incubation time 1. Antibody concentration too high 2. Over cell conditioning 1. Decrease antibody concentration and/or decrease antibody incubation time 2. Decrease or remove cell conditioning steps Intellectual Property UltraMap 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 , B1, 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 Technical Customer Care at: U.S.A. : (800) Ventana Medical Systems, Inc E. Innovation Park Drive Tucson, AZ USA Japan : (+81) Ventana Japan K.K. Landmark Tower, 35F 2-2-1, Minato Mirai, Nishi-ku Yokohama, Kanagawa Japan Europe : (+33) Ventana Medical Systems S.A. Parc d Innovation Rue G. de Kaysersberg BP F ILLKIRCH Cedex France Australia : +61 (0) Ventana Medical Systems, Pty. Ltd. 5/39 Grand Boulevard Montmorency, Victoria 3094 Australia Ventana Translational Diagnostics Group June