May 31, 2026
OPAL Multiplex Immunofluorescence (mIF) Protocol: 4-Plex Staining for CD155, GPNMB, CD31, and CD112
- Franz Zemp1,
- Hongrui Lui1,
- Douglas Mahoney1
- 1University of Calgary
Protocol Citation: Franz Zemp, Hongrui Lui, Douglas Mahoney 2026. OPAL Multiplex Immunofluorescence (mIF) Protocol: 4-Plex Staining for CD155, GPNMB, CD31, and CD112. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lymznmlx9/v1
Manuscript citation:
Zemp et al. 2026. GPNMB-directed CAR T-cell therapy against MiT/TFE family fusion-driven solid tumors. Nature Cancer. Accepted.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 27, 2026
Last Modified: May 31, 2026
Protocol Integer ID: 318071
Keywords: opal multiplex immunofluorescence, multiplex immunofluorescence, quantification of multiple protein target, plex staining for cd155, cd112 simultaneous detection, multiple protein target, plex staining, plex manual detection kit
Abstract
Simultaneous detection and quantification of multiple protein targets in formalin-fixed, paraffin-embedded (FFPE) tissue sections using the Opal 6-Plex Manual Detection Kit (Akoya Biosciences). This protocol enables multiplex immunofluorescence staining for CD155, GPNMB, CD31, and CD112 with tyramide signal amplification (TSA) and spectrally distinct fluorophores.
Guidelines
- All staining steps must strictly follow the manufacturer’s protocol for the Opal 6-Plex Manual Detection Kit.
- Antigen retrieval buffer choice is marker-specific; when performing multiplex staining, select the buffer that provides optimal performance for the entire panel (typically determined during assay optimization).
- Include appropriate positive and negative controls (e.g., autofluorescent slides, isotype controls or single-plex slides) in each run.
- Protect fluorophore-stained slides from light during and after staining to preserve signal intensity.
- Proper spectral unmixing is critical for accurate signal separation in multiplex imaging.
- This protocol is designed for manual staining; minor adjustments may be required for automated platforms.
Materials
- Adhesive Microscope slides (Fisher Scientific, 12-550-15)
- Xylene (histological grade)
- Ethanol (100%, 95%, 70%) for rehydration gradient
- Distilled water
- 10X AR6 buffer (Akoya Biosciences, AR600250ML) – for antigen retrieval of GPNMB and CD31
- 10X AR9 buffer (Akoya Biosciences, AR900250ML) – for antigen retrieval of CD155 and CD112
- Protein Block (Agilent, cat. no. X090930-2)
- Primary antibodies:
- CD155 (D8A5G, Cell Signaling, #81254, 1:100)
- GPNMB (E4D7P, Cell Signaling, #38313, 1:200)
- CD31 (Abcam, ab28364, 1:100)
- CD112 (D8D3F, Cell Signaling, #95333, 1:200)
- Opal 6-Plex Manual Detection Kit (Akoya Biosciences, cat. no. NEL811001KT) containing Opal fluorophores:
- Opal 520 (1:200)
- Opal 570 (1:200)
- Opal 690 (1:200)
- Opal 780 (1:25)
- Oven (set to 60°C)
- Pressure cooker (for heat-induced epitope retrieval)
- Humidified slide incubation chamber
- Slide racks and staining dishes
- Akoya Vectra Polaris imaging system
- HALO image analysis software (Indica Labs, v4.0.5107.407)
Procedure
Section Preparation. Cut FFPE tissue samples into 5 µm thick sections and mount onto charged slides.
Deparaffinization. Incubate slides for 3 hours at 60°C. Perform three washes in xylene, 5 minutes each.
Rehydration. Rehydrate sections through a graded ethanol series:
100% ethanol (2 changes, 5 minutes each)
95% ethanol (5 minutes)
70% ethanol (5 minutes)
Distilled water (5 minutes)
Heat-Induced Antigen Retrieval (HIER). Perform antigen retrieval in a pressure cooker for 22 minutes using the appropriate buffer:
1X AR6 buffer for GPNMB and CD31
1X AR9 buffer for CD155 and CD112
Allow slides to cool to room temperature in the buffer.
Rinse in distilled water followed by 1× TBST.
Non-Specific Binding Block. Apply 200 µL of Protein Block (Agilent, X090930-2) to each section. Incubate for 30 minutes at room temperature. Gently remove excess blocking solution (do not rinse).
Multiplex Immunofluorescence Staining. Perform sequential multiplex staining according to the manufacturer’s instructions for the Opal 6-Plex Manual Detection Kit (Akoya Biosciences). Apply primary antibodies and corresponding Opal fluorophores in the following order and repeat step 4-6 until all markers are stained.
CD155 (1:100) – incubate overnight at 4°C, paired with Opal 520 (1:200)
GPNMB (1:200) – incubate 1 hour at room temperature, paired with Opal 570 (1:200)
CD31 (1:100) – incubate overnight at 4°C, paired with Opal 690 (1:200)
CD112 (1:200) – incubate overnight at 4°C, paired with Opal 780 (1:25) Include appropriate stripping and washing steps between each antibody cycle as recommended by the Opal kit protocol.
Slide Scanning. Scan stained slides using the Akoya Vectra Polaris at 20× resolution.
Image Analysis. Import scanned images into HALO software (Indica Labs, v4.0.5107.407) for visualization, spectral unmixing, and quantitative analysis of marker expression, co-localization, and cellular phenotyping.