Immunohistochemistry (Leica®BOND™)
NOTE: Please see product datasheet or product webpage for appropriate antibody dilution^.
| 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 | 60 min. |
| WASH | BOND™ Wash Solution | 3x 2:00 min. | |
| 6 | Post Primary Mouse Linker | Refine Detection Kit Post Primary* | 10 min. |
| WASH | BOND™ Wash Solution | 3x 2:00 min. | |
| 7 | Secondary Detection | Refine Detection Kit Polymer* | 10 min. |
| WASH | BOND™ Wash Solution/Deionized Water | Custom (see below) | |
| 8a | Visualization | Refine Detection Kit Mixed DAB Refine* | 0:00 min. |
| 8b | Visualization | Refine Detection Kit Mixed DAB Refine* | 10 min. |
| WASH | Deionized Water | 3x 0:00 min. | |
| 9 | 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. | |
| 10 | 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. | |||
| 11 | Mount sections with coverslips and #84583 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 August 2018
revised September 2018