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
