Automated Multiplex IHC Staining with Aluora Spatial Amplification Kits and Leica BOND System

Introduction

This protocol outlines the steps for performing multiplexed staining of a tissue sample using a combination of purified primary antibodies and enzymatically mediated signal amplification assays. Two options for signal amplification are available: Invitrogen Aluora Spatial Amplification Kits and reagents or Invitrogen Alexa Fluor SuperBoost tyramide kits and reagents.

The use of signal amplification reagents is recommended for multiplexed imaging of tissue samples with low to medium expressed markers, especially when the signal obtained with antibody labeling alone is low. Aluora Spatial Amplification Kits can improve the signal-to-background ratio and boost signal. It is also beneficial for high abundance markers as it allows for reduced antibody usage.

For tissue samples and multiplexing multiple markers, we recommend utilizing the Aluora Spatial Amplification Kits and reagents. Tissues labeled with Aluora dyes are prepared for subsequent labeled-antibody or nuclei counterstaining steps, and can be imaged using any type of fluorescence microscope or spatial imager, including the Invitrogen EVOS S1000 Spatial Imaging System or Akoya PhenoImager.

It is important to note that this protocol assumes the availability of an automated slide stainer, such as the Leica BOND RX systems, to perform the required steps.

Materials

Primary antibodies to your targets specific for IHC. Learn more about our advanced verified antibodies
DAPI (Cat. No. D1306)
Aluora Spatial Amplification Kit, multiple options available
Mountant with antifade properties such as ProLong Glass (Ca. No. P36980) or SlowFade Glass (Cat. No. S36917)

Leica BOND bulk solutions including:

Leica Biosystems BOND WASH SOLUTION 10X (Cat. No. NC02221077)
Leica Biosystems BOND EPITOPE RETRIEVAL SOLUTION (Cat. No. NC0235529)
Leica Biosystems BOND OPEN CONTAINERS (Cat. No. NC0498827) —used for wash buffers and other reagents
Leica Biosystems TITRATION KIT (6mL) (Cat. No. NC209406) —used for antibodies and Invitrogen Aluora reagents
Leica Biosystems BOND UNIVERSAL COVERTILES (Cat. No. NC1206859)
Leica Biosystems BOND RESEARCH DETECTION SYSTEM (Cat. No. NC1312938)
Leica Biosystems BOND DEWAX SOLUTION (Cat. No. NC0221076)

Procedure

Procedural guidelines and tips

Crisp staining results require optimizing primary antibody dilution.
The Aluora dye staining order suggested here is just an example. Staining order may need to be optimized to achieve best results.
We recommend reading the Leica BOND instruction manual and training guide before starting your experiment. Contact the Leica customer service for support or questions on their instruments.
We recommend making Aluora reagents fresh each day.

Prepare controls

The following negative and single-color controls are required for optimal spectral unmixing of the multiplex targets. Prepare your controls using the same workflow and equipment as multiplex sample. Please prepare each of the controls including:

Single-color controls	Protocol
Single-color control: Make one for each primary antibody conjugate found in multiplex staining	Please follow the BOND protocol below.
	Fill one titration kit container with one Aluora reagent. Replace other titration kit containers with wash buffers instead of dye.
Nuclear counterstain only control	Add nuclear counterstain such as NucBlue or DAPI at the same concentration as was used in the multiplex sample. Replace other titration kit containers with wash buffers instead of stain.
Nuclear counterstain only control	Proceed to mounting step.
Unstained tissue sample	No counter stain or any fluorophore conjugates
Unstained tissue sample	Proceed to mounting step

We strongly recommend you optimize the primary antibody before doing the multiplex staining. Stain tissues with a serial dilution including 1:1,000 to 1:10,000 of the primary antibody conjugated to a fluorophore. If the target is highly expressed, some cases the dilution can be 1:50,000.

BOND system protocol set-up

Create BOND system protocol for multiple rounds of staining from cycle 1 – 8. Follow BOND system manual to enter steps manually.

Tissue preparation

In the drop-down menu select pre-set preparation protocol. This should be based on your tissue type and needs. The step will include:

Deparaffinization and rehydration

Temperature: 72°C

30:00

150 μL

Enter BOND system cycles

BOND settings for staining cycles

Use the following settings for up to 8 staining cycles on the Leica BOND system.

Step	Reagent	Temp	Incubation	Dispense	Staining cycle
1	*Bond Wash Solution	Ambient	0:00	150 μL
2	*Bond Wash Solution	Ambient	0:00	150 μL
3	Antibody Block	Ambient	5:00	150 μL	1
4	Antibody 1	Ambient	30:00	150 μL
5	*Bond Wash Solution	Ambient	0:00	150 μL
6	*Bond Wash Solution	Ambient	1:00	150 μL
7	*Bond Wash Solution	Ambient	0:00	150 μL
8	HRP secondary Ab	Ambient	10:00	150 μL
9	*Bond Wash Solution	Ambient	0:00	150 μL
10	*Bond Wash Solution	Ambient	1:00	150 μL
11	*Bond Wash Solution	Ambient	0:00	Open
12	*Bond Wash Solution	Ambient	0:00	150 μL
13	*Bond Wash Solution	Ambient	0:00	150 μL
14	Aluora 430 dye	Ambient	10:00	150 μL
15	*Bond Wash Solution	Ambient	0:00	150 μL
16	*Bond Wash Solution	Ambient	1:00	150 μL
17	*Bond Wash Solution	Ambient	0:00	150 μL
18	*Bond Wash Solution	Ambient	0:00	150 μL
19	*Bond ER Solution 1	Ambient	0:00	150 μL
20	*Bond ER Solution 1	95°C	0:00	150 μL
21	*Bond ER Solution 1	95°C	20:00	Intermediate
22	*Bond ER Solution 1	Ambient	0:00	150 μL
23	*Bond Wash Solution	Ambient	0:00	150 μL
24	*Bond Wash Solution	Ambient	1:00	150 μL
25	*Bond Wash Solution	Ambient	0:00	150 μL
26	*Bond Wash Solution	Ambient	0:00	150 μL
27	*Bond Wash Solution	Ambient	0:00	150 μL
28	Antibody Block	Ambient	5:00	150 μL	2
29	Antibody 2	Ambient	30:00	150 μL
30	*Bond Wash Solution	Ambient	0:00	150 μL
31	*Bond Wash Solution	Ambient	1:00	150 μL
32	*Bond Wash Solution	Ambient	0:00	150 μL
33	HRP secondary Ab	Ambient	10:00	150 μL
34	*Bond Wash Solution	Ambient	0:00	150 μL
35	*Bond Wash Solution	Ambient	1:00	150 μL
36	*Bond Wash Solution	Ambient	0:00	Open
37	*Bond Wash Solution	Ambient	0:00	150 μL
38	*Bond Wash Solution	Ambient	0:00	150 μL
39	Aluora 488 dye	Ambient	10:00	150 μL
40	*Bond Wash Solution	Ambient	0:00	150 μL
41	*Bond Wash Solution	Ambient	1:00	150 μL
42	*Bond Wash Solution	Ambient	0:00	150 μL
43	*Bond Wash Solution	Ambient	0:00	150 μL
44	*Bond ER Solution 1	Ambient	0:00	150 μL
45	*Bond ER Solution 1	95°C	0:00	150 μL
46	*Bond ER Solution 1	95°C	20:00	Intermediate
47	*Bond ER Solution 1	Ambient	0:00	150 μL
48	*Bond Wash Solution	Ambient	0:00	150 μL
49	*Bond Wash Solution	Ambient	1:00	150 μL
50	*Bond Wash Solution	Ambient	0:00	150 μL
51	*Bond Wash Solution	Ambient	0:00	150 μL
52	*Bond Wash Solution	Ambient	0:00	150 μL
53	Antibody Block	Ambient	5:00	150 μL	3
54	Antibody 3	Ambient	30:00	150 μL
55	*Bond Wash Solution	Ambient	0:00	150 μL
56	*Bond Wash Solution	Ambient	1:00	150 μL
57	*Bond Wash Solution	Ambient	0:00	150 μL
58	HRP secondary Ab	Ambient	10:00	150 μL
59	*Bond Wash Solution	Ambient	0:00	150 μL
60	*Bond Wash Solution	Ambient	1:00	150 μL
61	*Bond Wash Solution	Ambient	0:00	Open
62	*Bond Wash Solution	Ambient	0:00	150 μL
63	*Bond Wash Solution	Ambient	0:00	150 μL
64	Aluora 514 dye	Ambient	10:00	150 μL
65	*Bond Wash Solution	Ambient	0:00	150 μL
66	*Bond Wash Solution	Ambient	1:00	150 μL
67	*Bond Wash Solution	Ambient	0:00	150 μL
68	*Bond Wash Solution	Ambient	0:00	150 μL
69	*Bond ER Solution 1	Ambient	0:00	150 μL
70	*Bond ER Solution 1	95°C	0:00	150 μL
71	*Bond ER Solution 1	95°C	20:00	Intermediate
72	*Bond ER Solution 1	Ambient	0:00	150 μL
73	*Bond Wash Solution	Ambient	0:00	150 μL
74	*Bond Wash Solution	Ambient	1:00	150 μL
75	*Bond Wash Solution	Ambient	0:00	150 μL
76	*Bond Wash Solution	Ambient	0:00	150 μL
77	*Bond Wash Solution	Ambient	0:00	150 μL
78	Antibody Block	Ambient	5:00	150 μL	4
79	Antibody 4	Ambient	30:00	150 μL
80	*Bond Wash Solution	Ambient	0:00	150 μL
81	*Bond Wash Solution	Ambient	1:00	150 μL
82	*Bond Wash Solution	Ambient	0:00	150 μL
83	HRP secondary Ab	Ambient	10:00	150 μL
84	*Bond Wash Solution	Ambient	0:00	150 μL
85	*Bond Wash Solution	Ambient	1:00	150 μL
86	*Bond Wash Solution	Ambient	0:00	Open
87	*Bond Wash Solution	Ambient	0:00	150 μL
88	*Bond Wash Solution	Ambient	0:00	150 μL
89	Aluora 555 dye	Ambient	10:00	150 μL
90	*Bond Wash Solution	Ambient	0:00	150 μL
91	*Bond Wash Solution	Ambient	1:00	150 μL
92	*Bond Wash Solution	Ambient	0:00	150 μL
93	*Bond Wash Solution	Ambient	0:00	150 μL
94	*Bond ER Solution 1	Ambient	0:00	150 μL
95	*Bond ER Solution 1	95°C	0:00	150 μL
96	*Bond ER Solution 1	95°C	20:00	Intermediate
97	*Bond ER Solution 1	Ambient	0:00	150 μL
98	*Bond Wash Solution	Ambient	0:00	150 μL
99	*Bond Wash Solution	Ambient	1:00	150 μL
100	*Bond Wash Solution	Ambient	0:00	150 μL
101	*Bond Wash Solution	Ambient	0:00	150 μL
102	*Bond Wash Solution	Ambient	0:00	150 μL
103	Antibody Block	Ambient	5:00	150 μL	5
104	Antibody 5	Ambient	30:00	150 μL
105	*Bond Wash Solution	Ambient	0:00	150 μL
106	*Bond Wash Solution	Ambient	1:00	150 μL
107	*Bond Wash Solution	Ambient	0:00	150 μL
108	HRP secondary Ab	Ambient	10:00	150 μL
109	*Bond Wash Solution	Ambient	0:00	150 μL
110	*Bond Wash Solution	Ambient	1:00	150 μL
111	*Bond Wash Solution	Ambient	0:00	Open
112	*Bond Wash Solution	Ambient	0:00	150 μL
113	*Bond Wash Solution	Ambient	0:00	150 μL
114	Aluora 594 dye	Ambient	10:00	150 μL
115	*Bond Wash Solution	Ambient	0:00	150 μL
116	*Bond Wash Solution	Ambient	1:00	150 μL
117	*Bond Wash Solution	Ambient	0:00	150 μL
118	*Bond Wash Solution	Ambient	0:00	150 μL
119	*Bond ER Solution 1	Ambient	0:00	150 μL
120	*Bond ER Solution 1	95°C	0:00	150 μL
121	*Bond ER Solution 1	95°C	20:00	Intermediate
122	*Bond ER Solution 1	Ambient	0:00	150 μL
123	*Bond Wash Solution	Ambient	0:00	150 μL
124	*Bond Wash Solution	Ambient	1:00	150 μL
125	*Bond Wash Solution	Ambient	0:00	150 μL
126	*Bond Wash Solution	Ambient	0:00	150 μL
127	*Bond Wash Solution	Ambient	0:00	150 μL
128	Antibody Block	Ambient	5:00	150 μL	6
129	Antibody 6	Ambient	30:00	150 μL
130	*Bond Wash Solution	Ambient	0:00	150 μL
131	*Bond Wash Solution	Ambient	1:00	150 μL
132	*Bond Wash Solution	Ambient	0:00	150 μL
133	HRP secondary Ab	Ambient	10:00	150 μL
134	*Bond Wash Solution	Ambient	0:00	150 μL
135	*Bond Wash Solution	Ambient	1:00	150 μL
136	*Bond Wash Solution	Ambient	0:00	Open
137	*Bond Wash Solution	Ambient	0:00	150 μL
138	*Bond Wash Solution	Ambient	0:00	150 μL
139	Aluora 647 dye	Ambient	10:00	150 μL
140	*Bond Wash Solution	Ambient	0:00	150 μL
141	*Bond Wash Solution	Ambient	1:00	150 μL
142	*Bond Wash Solution	Ambient	0:00	150 μL
143	*Bond Wash Solution	Ambient	0:00	150 μL
144	*Bond ER Solution 1	Ambient	0:00	150 μL
145	*Bond ER Solution 1	95°C	0:00	150 μL
146	*Bond ER Solution 1	95°C	20:00	Intermediate
147	*Bond ER Solution 1	Ambient	0:00	150 μL
148	*Bond Wash Solution	Ambient	0:00	150 μL
149	*Bond Wash Solution	Ambient	1:00	150 μL
150	*Bond Wash Solution	Ambient	0:00	150 μL
151	*Bond Wash Solution	Ambient	0:00	150 μL
152	*Bond Wash Solution	Ambient	0:00	150 μL
153	Antibody Block	Ambient	5:00	150 μL	7
154	Antibody 7	Ambient	30:00	150 μL
155	*Bond Wash Solution	Ambient	0:00	150 μL
156	*Bond Wash Solution	Ambient	1:00	150 μL
157	*Bond Wash Solution	Ambient	0:00	150 μL
158	HRP secondary Ab	Ambient	10:00	150 μL
159	*Bond Wash Solution	Ambient	0:00	150 μL
160	*Bond Wash Solution	Ambient	1:00	150 μL
161	*Bond Wash Solution	Ambient	0:00	Open
162	*Bond Wash Solution	Ambient	0:00	150 μL
163	*Bond Wash Solution	Ambient	0:00	150 μL
164	Aluora 700 dye	Ambient	10:00	150 μL
165	*Bond Wash Solution	Ambient	0:00	150 μL
166	*Bond Wash Solution	Ambient	1:00	150 μL
167	*Bond Wash Solution	Ambient	0:00	150 μL
168	*Bond Wash Solution	Ambient	0:00	150 μL
169	*Bond ER Solution 1	Ambient	0:00	150 μL
170	*Bond ER Solution 1	95°C	0:00	150 μL
171	*Bond ER Solution 1	95°C	20:00	Intermediate
172	*Bond ER Solution 1	Ambient	0:00	150 μL
173	*Bond Wash Solution	Ambient	0:00	150 μL
174	*Bond Wash Solution	Ambient	1:00	150 μL
175	*Bond Wash Solution	Ambient	0:00	150 μL
176	*Bond Wash Solution	Ambient	0:00	150 μL
177	*Bond Wash Solution	Ambient	0:00	150 μL
178	Antibody Block	Ambient	5:00	150 μL	8
179	Antibody 8	Ambient	30:00	150 μL
180	*Bond Wash Solution	Ambient	0:00	150 μL
181	*Bond Wash Solution	Ambient	1:00	150 μL
182	*Bond Wash Solution	Ambient	0:00	150 μL
183	HRP secondary Ab	Ambient	10:00	150 μL
184	*Bond Wash Solution	Ambient	0:00	150 μL
185	*Bond Wash Solution	Ambient	1:00	150 μL
186	*Bond Wash Solution	Ambient	0:00	Open
187	*Bond Wash Solution	Ambient	0:00	150 μL
188	*Bond Wash Solution	Ambient	0:00	150 μL
189	Aluora 750 dye	Ambient	10:00	150 μL
190	*Bond Wash Solution	Ambient	0:00	150 μL
191	*Bond Wash Solution	Ambient	1:00	150 μL
192	*Bond Wash Solution	Ambient	0:00	150 μL
193	*Bond Wash Solution	Ambient	0:00	150 μL
194	*Bond ER Solution 1	Ambient	0:00	150 μL
195	*Bond ER Solution 1	95°C	0:00	150 μL
196	*Bond ER Solution 1	95°C	20:00	Intermediate
197	*Bond ER Solution 1	Ambient	0:00	150 μL
198	*Bond Wash Solution	Ambient	0:00	150 μL
199	*Bond Wash Solution	Ambient	1:00	150 μL
200	*Bond Wash Solution	Ambient	0:00	150 μL
201	DAPI	Ambient	5:00	150 μL
202	*Bond Wash Solution	Ambient	0:00	150 μL
203	*Bond Wash Solution	Ambient	1:00	150 μL
204	*Bond Wash Solution	Ambient	0:00	150 μL

Reagent preparation

Dilute primary antibodies and make secondary antibody mix with equal volumes Poly-HRP-Goat Anti-Mouse IgG+ Poly-HRP-Goat Anti-Rabbit IgG. If using biotinylated primaries, include a biotin/strep-avidin blocking steps in that staining cycle and use the HRP-strepavidin reagent instead of poly-HRP antibody. Make up Aluora dye staining solution according to Aluora Spatial Amplification Kit user guide.

Prepare reagents for instrument:
- Prepare the antibody and the fluorophore dilutions in 6 mL Leica BOND tubes found in the titration kit.
  - The minimum amount of reagent or liquid in tube is 300 µL. Add 100 µL or 150 µL of antibodies or Aluora reagents depending on selection in BOND workflow.
  - Fill an empty 6 mL Leica BOND container with 1x DAPI.
  - Fill an empty 6 mL Leica BOND container with block reagent.
Add wash buffers into the appropriate container.

Follow the Leica BOND system protocol to run assay

Run the programmed staining cycles. If additional support is needed, contact the Leica customer service.

Mounting

Mount the coverslips using a mountant with antifade properties:

ProLong Glass Antifade Mountant (Cat. No. P36980) is recommended for hard-set mounting and long-term archiving of tissue samples.
SlowFade Glass Soft-set Antifade Mountant (Cat. No. S36917) is recommended for soft-set mounting if coverslip removal is required for tissue samples, and the slide is being imaged from above.
For optimal results, follow the instructions provided with the mountant.

Analyze the tissue using a compatible imaging instrument.

PhenoImager is trademarked by Akoya Biosciences. BOND are trademarks of Leica Biosystems and its affiliates.

Invitrogen EVOS S1000 can be purchased in participating countries and regions including US, Canada, and Europe for 2024.

For Research Use Only. Not for use in diagnostic procedures.