After CAR-T infusion (B7-H3 CAR-T cells or control CD19 CAR-T cells) on day 9, tumors were resected from the NBEB s.c. mice and fixed with 10% neutral buffered formalin and processed for routine hematoxylin and eosin (H&E) staining was conducted by Histoserv, Inc (Germantown, MD). Immunofluorescence and immunohistochemistry staining was performed by the Molecular Histopathology Laboratory (MHL) at the NCI. Briefly, after antigen retrieval with EDTA (Bond Epitope Retrieval 2) and endogenous peroxidase blocking, sections were incubated with anti-CD3 mAb (Bio-Rad, #MCA1477), followed by OPAL Fluorophore 520 (AKOYA). The anti-CD3 mAb complex was stripped by heating with Bond Epitope Retrieval 2. Sections were then incubated with anti-B7-H3 mAb (clone D9M2L, Cell Signaling Technology, #14058) followed by Bond Polymer reagent and OPAL Fluorophore 690 (AKOYA). Sections were removed from the Bond, DAPI stained, and coverslipped with Prolong Gold AntiFade Reagent (Invitrogen). Cleaved Caspase-3 (Cell Signaling Technology, #9661) immunohistochemistry was performed to predict cell apoptosis. Images were captured using the Aperio Scanscope FL (or XT) whole slide scanner, and staining was interpreted by a board-certified veterinary pathologist.
Opal 520 fluorophore
Manufactured by Akoya Biosciences
Sourced in United States
The Opal 520 Fluorophore is a fluorescent dye used in immunohistochemistry (IHC) and multiplex assays. It emits green fluorescence when excited by a compatible light source.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Halo imaging analysis software, by Indica Labs (1 mentions)
Bond polymer refine kit, by Leica (1 mentions)
Ab28364, by Thermo Fisher Scientific (1 mentions)
At2 scanner, by Leica (1 mentions)
Bond rx, by Leica (1 mentions)
Opal 570, by Akoya Biosciences (1 mentions)
Opal 690, by Akoya Biosciences (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Gfp 1020, by Abcam (1 mentions)
Ab53554, by Fujifilm (1 mentions)
Ab4055, by Abcam (1 mentions)
Tyramide signal amplification, by Akoya Biosciences (1 mentions)
Bz x710 all in one fluorescence microscope, by Keyence (1 mentions)
N21479, by Thermo Fisher Scientific (1 mentions)
Cyanine 3, by Jackson ImmunoResearch (1 mentions)
Anti cd31 antibody, by BD (1 mentions)
Cyanine 5, by Jackson ImmunoResearch (1 mentions)
7 protocols using opal 520 fluorophore
1
Immunofluorescence Analysis of B7-H3 CAR-T Therapy
2
Brain Vasculature Imaging and Analysis
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Fourteen days after tumor implantation, 6 mg/kg of Texas Red-conjugated 3 kDa dextran (TRD, ThermoFisher) was injected intravenously through the tail vein. TRD was allowed to circulate for 20 min, animals were exsanguinated by intracardiac perfusion of PBS with subsequent perfusion of 4% PFA solution for brain tissue fixation. Extracted brains were embedded in paraffin and sectioned at 5 μm thickness. Formalin-fixed sections were then subjected to either TRD staining or immunohistochemical (IHC) staining. Whole slide images were obtained at high resolution, 20×g using an AT2 scanner (Aperio, Leica Biosystems, Buffalo Grove, IL). Double staining for CD31/Claudin5 were performed on the Leica Biosystems Bond RX autostainer using the Bond Polymer Refine Kit (Leica Biosystems DS9800), with omission of the Post Primary reagent, DAB and Hematoxylin. Slides were double stained for CD31 (Abcam ab28364, 1:100), and Claudin-5 (Invitrogen 35-2500, 1:50). Images were captured using the Aperio Scanscope FL whole slide scanner (Leica Biosystems) into whole slide digital images and OPAL Fluorophore 520 (AKOYA) as per previously published [25 (link)]. Image analysis was performed using Halo imaging analysis software (v3.3.2541.423, Indica Lab’s, Corrales, NM) using Halo algorithm (Area Quantification FL v2.3.4) which were used to quantify positive area of TRD, CD31, and Claudin5.
3
Multiplexed RNA in situ Hybridization
4
Multi-target RNA In Situ Hybridization
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RNA in situ hybridization was performed using RNAscope® Multiplex Fluorescent Reagent Kit (ACD, 323110) according to the manufacturer’s instructions. Briefly, brain sections were fixed in 4% paraformaldehyde at 4 °C for 15 min, ethanol-dehydrated (Carl Roth #9065.4), treated with H2O2 (ACD, 322381) and protease-permeabilized for 20 min at 40 °C. Brain sections were then incubated for 2 h at 40 °C using the following probes: Ifi27l2a (ACD, 88617), Serpina3n (ACD, 430191-C2), Lcn2 (ACD, 313971-C3), Cd68 (ACD, 316611-C2), Oasl2 (ACD, 534501), and Cxcl10 (ACD, 408921-C3). Signal was amplified according to the manufacturer’s instructions (User manual Cat.Nr: 320293, Fluorophore Opal 520: Akoya Biosciences FP1488001KT). Subsequently, sections were processed with immunohistochemistry analysis as described above. The primary antibodies used in combination with RNAscope® were as follows: chick antibody to GFP (1:400, Aves Lab, GFP-1020), goat antibody to GFAP (1:300, Abcam, ab53554), rabbit antibody to IBA1 (1:500, Wako, 019-19741).
5
Multiplexed Immunofluorescence Analysis of HLA-I and CD8
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The sections were prepared, and endogenous peroxidase was blocked as described above. Nonspecific binding sites were blocked by incubation with Protein Block Serum-Free Reagent for 30 min, and the sections were incubated overnight at 4 °C with the primary antibodies against HLA Class I-A, B, C (Hokudo, Sapporo, Japan, EMR8-5, mouse mAb, 1:400 dilution) and CD8 (Abcam, Cambridge, MA, USA, ab4055, rabbit pAb, 1:400 dilution). Multiplex covalent labeling (HLA-I, Opal 520 Fluorophore, OP-001001; CD8, Opal 570 Fluorophore, OP-001003) with tyramide signal amplification (Akoya Biosciences, MA, USA) was performed according to the manufacturer’s protocol. All sections were counterstained with DAPI and examined under an All-in-One BZ-X710 fluorescence microscope (KEYENCE Corporation, Osaka, Japan).
6
RNAscope-Based Multiplex Fluorescent Imaging
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The RNAscope TM Multiplex Fluorescent V2 kit (Biotechne, MN, USA) was used according to the manufacturer's instructions. All laboratory solutions were prepared in advance with autoclaved water at 0.1% DEPC (Sigma-Aldrich, MO, USA; D5758) to avoid any contamination by RNAses and DNAses. Our target was revealed with the Plat probe, revealing the plat gene encoding tPA (Biotechne, MN, USA; 586951) coupled with Opal 520 fluorophore (Akoya, MA, USA; SKU FP1487001KT; 1/5000). At the end of the protocol, we carried out immunostaining steps with the primary antibodies incubated overnight at 4 °C (anti-CD31 antibody, BD Biosciences; 555024 1/500; anti-phalloidin antibody, Invitrogen N21479 1/300). After 3 washes in 1 × PBS, the appropriate secondary antibodies coupled with Cyanine 3 or Cyanine 5 (1/800 Jackson Immunoresearch, West Grove, CA, USA) were incubated at room temperature for 90 min. Once washed, the slides were mounted with coverslip and mounting medium with DAPI (Fluoromont-G; Thermofisher, MA USA; 15596276).
7
Multicolor Immunofluorescence Staining Protocol
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The sections were labeled with an IFN-γ polyclonal antibody (ImmunoWay, Suzhou, China. cat# YT2279) at a 1:500 dilution for 1 h at room temperature and visualized with an Opal 520 Fluorophore (Akoya Biosciences, Massachusetts, U.S.A. Opal 3-Plex Manual Detection Kit, REF: NEL810001KT). Antigen retrieval was subsequently performed. The tissue sections were blocked for 1 h in 5% BSA at room temperature and then labeled with the Rb pAb to IL-1 (Abcam, ab2105) at a 1:500 dilution overnight at 4 °C. Finally, the IL-1 protein antigens were visualized via incubation with donkey anti-rabbit IgG H&L (Alexa Fluor® 594) (Abcam, ab150076) at a 1:500 dilution for one hour. The images were captured via a Leica TCS SP8 laser confocal microscope.
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