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 1X PBST (9809)	30 min.
	WASH	BOND™ Wash Solution	3x 2:00 min.
NA	Post Primary Mouse Linker	Refine Detection Kit Post Primary*	Not Applied
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
	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.