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 #84583 SignalStain® Mounting Medium II
	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)

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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.