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Immunohistochemistry Leica® BOND™ Protocol

IMPORTANT: Please refer to the APPLICATIONS section on the front page of the product datasheet or product webpage to determine whether a product is validated and approved for use on the Leica® BOND™. If so, refer to the product-specific protocol for instructions. For products without a validated Leica® BOND™ protocol, the conditions listed below can serve as a starting point.

  Step Reagents Time/Temperature
1 Dewax BOND™ Dewax Solution, 100% Alcohol, BOND™ Wash Solution Pre-programmed Leica® BOND™
2 Antigen Retrieval BOND™ Epitope Retrieval ER2 Solution 20 min., 100˚C | Protocol: HIER 20 min with ER2
3 Peroxide Block Refine Detection Kit Peroxide Block* 5 min.
  WASH BOND™ Wash Solution 3x 0:00 min.
4 Protein Block (optional) #5425 NGS or #15019 Animal-Free Blocking Solution 20 min.
5 Primary Antibody^ Dilute in #8112 SignalStain® Antibody Diluent 30 min.
  WASH BOND™ Wash Solution 3x 2:00 min.
NA Post Primary Mouse Linker Refine Detection Kit Post Primary* Not applied for rabbit antibodies, 10 min. for mouse antibodies
6 Secondary Detection Refine Detection Kit Polymer* 10 min.
  WASH BOND™ Wash Solution/Deionized Water Custom (see below)
7a Visualization Refine Detection Kit Mixed DAB Refine* 0:00 min.
7b Visualization Refine Detection Kit Mixed DAB Refine* 10 min.
  WASH Deionized Water 3x 0:00 min.
8 Counterstain Refine Detection Kit Hematoxylin* 5 min.
  WASH Deionized Water 0:00 min.
  WASH BOND™ Wash Solution 0:00 min.
  WASH Deionized Water 0:00 min.
9 Dehydration (Offline):    
  Incubate sections in 95% ethanol two times for 10 seconds each.  
  Repeat in 100% ethanol, incubating sections two times for 10 seconds each.  
  Repeat in xylene, incubating sections two times for 10 seconds each.  
10 Mount sections with coverslips and #14177 SignalStain® Mounting Medium  
       
  Optional Custom wash: BOND™ Wash Solution 2:00
    BOND™ Wash Solution Dispenser Type: OPEN 0:00
    BOND™ Wash Solution 2:00
    BOND™ Wash Solution Dispenser Type: OPEN 0:00
    BOND™ Wash Solution 0:00
    Deionized Water 0:00

*Reagent included in BOND™ Polymer Refine Detection Kit (Catalog No: DS9800)

LEICA® is a registered trademark of Leica Microsystems IR GmbH.

BOND™ is a trademark of Leica Biosystems Melbourne Pty. Ltd. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty. Ltd is implied.

posted July 2019

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