Rabbit monoclonal antibodies to CD4 (Cell Marque, Rocklin, CA) clone 104R-1, 1/100, high pH retrieval), mouse monoclonal antibody to FOXP3 (Abcam, Cambridge, UK), clone IgG1, 1/1000, high pH retrieval), mouse polyclonal antibody to PD-1 (Abcam, IgG1 Nat105, 1/500, low pH retrieval), mouse monoclonal antibody to CTLA-4 (MyBiosource, San Diego, CA) IgG2a/k, 1/100, high pH retrieval) were used. Antibodies were diluted using a background-reducing antibody diluent buffer S3022 (Agilent, Santa Clara, CA). A horseradish hydrogen peroxidase (HRP) linked anti-mouse and anti-rabbit secondary antibody, EnvisionTM HRP (Agilent), was used for each primary antibody species according to the manufacturer’s recommendation. Immunofluorescent signal was visualized using the TSA amplification system, OPAL™ 7-color fluorescent IHC kit (Perkin Elmer), TSA dyes 540, 620, 650, and 690 (1:50) for ten minutes, counterstained with Spectral DAPI. All slides were imaged on the Vectra® 3 Quantitative Pathology Imaging System (Perkin Elmer). Images were then examined using color separation and inForm® Software v2.1 (Perkin Elmer). All slides were scanned on the Vectra at 10× magnification in order to select for high-powered imaging at 20× (resolution of 0.5 μm per pixel) using Phenochart (Perkin Elmer).
Opal 7 color fluorescent ihc kit
Manufactured by PerkinElmer
The OPAL™ 7-color fluorescent IHC kit is a laboratory equipment product that enables simultaneous detection of up to seven protein targets in a single tissue sample using fluorescent immunohistochemistry (IHC) techniques. The kit provides the necessary reagents and protocols to perform multiplexed fluorescent IHC analysis.
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Lab products found in correlation
Vectra 3 quantitative pathology imaging system, by PerkinElmer (1 mentions)
Phenochart, by PerkinElmer (1 mentions)
S3022, by Agilent Technologies (1 mentions)
Ab133616, by Abcam (1 mentions)
Ab16669, by Abcam (1 mentions)
Ab197678, by Abcam (1 mentions)
Ab783, by Abcam (1 mentions)
Ab104139, by Abcam (1 mentions)
Inform image analysis software, by PerkinElmer (1 mentions)
Hluga180su08, by Shanghai Outdo Biotech Co. (1 mentions)
Trilogy buffer, by Cell Marque (1 mentions)
Bsa pbs, by Merck Group (1 mentions)
4 protocols using opal 7 color fluorescent ihc kit
1
Multiparametric Immunofluorescence Staining
2
Multiplex IHC Analysis of Tissue Microarrays
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We constructed tissue microarrays as previously described13 using 2 tumor and 2 non‐tumor core specimens from each patient, each with a diameter of 1.5 mm. For multiplex IHC analysis of the microarrays, we used the Opal 7‐Color Fluorescent IHC Kit in accordance with the manufacturer's protocol (NEL811001KT; PerkinElmer) with the following fluorescent markers: DAPI (1:100; ab104139; abcam), anti‐CD3 (1:100; ab16669; abcam), anti‐CD4 (1:500; ab133616; abcam), anti‐CD8 (1:200; #85336; CST), anti‐CD66b (1:100; ab197678; abcam), and anti‐CD68 (1:100; ab783; abcam). Opal multiplexing is a serial IHC method that relies on tyramide single amplification. Opal slides can be visualized using standard fluorescence microscopy. Multispectral imaging allows quantitative unmixing of various fluorophores and removal of tissue autofluorescence. We analyzed fluorescence microscopy images using Inform image analysis software (version 2.4; PerkinElmer). All representative images were chosen by 3 pathologists.
3
Multiplex Immunofluorescence Staining of Lung Cancer Tissue
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We purchased a lung cancer tissue array (HLugA180Su08) from Shanghai Outdo Biotech and performed multiplex immunofluorescence staining (mIHC) using an Opal 7-color fluorescent IHC kit (Catlog Number: NEL801001KT, PerkinElmer) to assess the expression of CD4, CD8, and LRRC3B. Primary antibodies to the following antigens were used: CD4 (ready to use, Abcarta, code number: PA285, monoclonal antibody), CD8 (ready to use, Abcarta, code number: PA067, monoclonal antibody), and LRRC3B (1:200, Affinity Biosciences, code number: DF16048, polyclonal antibody). The primary antibodies were incubated at room temperature for 1h, followed by incubation with secondary antibodies. Nuclei were stained with DAPI. During the process of the mIHC, we first performed pre-experimental optimization of immunohistochemistry and then the polychromatic pre-experiment was performed twice. Fluorescence images were acquired using the TissueFAXS Views software (http://www.jsstl.cn/ ).
4
Multicolor Fluorescent IHC of ccRCC
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Formalin-fixed paraffin embedded sections from M-5 positive and control ccRCC samples were stained using the Opal 7-Color Fluorescent IHC Kit (PerkinElmer) according to manufacturer’s protocol. Slides were deparaffinized and rehydrated and antigen retrieved using Trilogy buffer (CellMarque) by autoclaving for 15 min. Slides were treated with 3% H2O2 for 15 min, washed, and blocked using 4% BSA/PBS/0.1% Triton X-100 (all from Sigma). Primary antibodies and consecutive HRP-conjugated secondary antibodies (Table S7 ) were diluted in 1% BSA/PBS/0.1% Triton X-100 and incubated for 1 hr at room temperature. Slides were then incubated in Amplification diluent containing a tyramide-conjugated fluorophore for 10 min. Prior to the second primary antibody incubation, the slides were heated for 10 min in 10 mM citric acid, pH 6.0 at 95°C to strip the antibodies of the first staining round. The protocol was repeated from the blocking step 3 to 4 times to co-stain several markers.
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