The Duolink in situ proximity ligation assay (PLA; OLINK Bioscience, Uppsala, Sweden) was utilized to detect the formation of AT1R/μOR heterodimers. The NTSs of SHRs and WKY were examined to detect the formation of AT1R (sc-515884) and μOR (bs-3623R) heterodimers in situ. The rats were first perfused in saline, then 4% formaldehyde, and finally 30% sucrose solution. Brainstem’s microsections of 5 μm thickness were obtained. Primary antibody diluent (OLINK Bioscience), containing two primary antibodies (1:100 for goat anti-μ receptor antibodies and 1:100 rabbit anti-AT1R antibodies), was added to the sections, and incubated overnight at 4 °C. Next, PLA secondary antibody with specific oligonucleotides, anti-rabbit plus and anti-goat minus (OLINK Bioscience), were applied to the sections and incubated for 2 h at 37 °C. Samples underwent ligation to allow nearby oligonucleotide probe pairs to form closed circles, and signals were amplified in the amplification solution. Images were acquired using a confocal laser scanning microscope (Carl Zeiss LSM 5 PASCAL), and were further processed by LSM 5 PASCAL software (Version 3.5, Carl Zeiss), which automatically counted the number of spots per unit of surface area.
Duolink in situ proximity ligation assay pla
Manufactured by Olink
Sourced in Sweden
Duolink in situ proximity ligation assay (PLA) is a laboratory technique used to detect and visualize protein-protein interactions in fixed cells or tissues. It utilizes a pair of oligonucleotide-labeled antibodies that bind to the target proteins, and when the proteins are in close proximity, the oligonucleotides can be joined, amplified, and detected as a fluorescent signal.
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Lab products found in correlation
Lsm 5 pascal, by Zeiss (1 mentions)
Lsm 5 pascal software, by Zeiss (1 mentions)
Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions)
Duolink in situ detection reagents orange, by Olink (1 mentions)
Tnt t7 coupled reticulocyte lysate system, by Promega (1 mentions)
Novolink polymer detection kit, by Leica (1 mentions)
Axioplan fluorescent microscope, by Zeiss (1 mentions)
Confocal lsm510 microscope, by Zeiss (1 mentions)
Blocking solution, by Olink (1 mentions)
Pab20849, by Abnova (1 mentions)
Nb100 58799, by Novus Biologicals (1 mentions)
Axiocam camera, by Zeiss (1 mentions)
Sc 9446, by Santa Cruz Biotechnology (1 mentions)
6 protocols using duolink in situ proximity ligation assay pla
1
Detecting AT1R/μOR Heterodimers in Rat Brain
2
Proximity Ligation Assay for TGFβ1 Signaling
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Human recombinant TGFβ1 was from Peprotech; LY2109761 was purchased from Selleckbio. Duolink in situ Proximity Ligation Assay (PLA) was from O-link Bioscience (Duolink In Situ Detection Reagents Orange), and was performed according to the manufacturer’s directions.
Antibodies used in the study are described in Supplementary Table4 (primary antibodies) and Supplementary Table 5 (secondary antibodies).
Antibodies used in the study are described in Supplementary Table
3
Detecting PD-L1 and p-EGFR Interactions via Duolink PLA
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Duolink in situ proximity ligation assay (PLA; Olink Bioscience) was used to detect the interactions of PD-L1 and p-EGFR (23 (link)). Immunofluorescence was performed as previously described (22 (link)). Oligonucleotide-conjugated PLA probe antibodies were directed against the primary antibodies for PD-L1 and p-EGFR. Annealing of the PLA probe occurred when PD-L1 and p-EGFR were in close proximity, which initiates the amplification of repeat sequences recognized by the fluorescently labeled oligonucleotide probe. For detection, Duolink detection kit 563 was used. The samples were imaged via confocal fluorescence microscopy (FV1000S-SIM/IX81, Japan).
4
Investigating p53 Interactions with PBF Using In Vitro Assays
5
Multimodal Microscopy Techniques for Tissue Analysis
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Immunofluorescence was performed as described previously (21 (link)). A Zeiss confocal LSM 510 microscope was used to perform confocal microscopy. Images were processed with ImageJ (http://imagej.net/Coloc_2 ) to determine Pearson's colocalization coefficients (39 (link)). Epifluorescent microscopy was performed on a Zeiss Axioplan fluorescent microscope. Human thyroid tissue was immunostained using the Novolink polymer detection kit (Leica) as per manufacturer's instructions. Slides were viewed under a light microscope (Zeiss) and images captured using Axiovision software. The Duolink in situ proximity ligation assay (PLA) was performed according to manufacturer's instructions (Olink Bioscience).
6
Detection of SPEN/MTA1 Protein Interactions
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To detect the SPEN/MTA1 interactions, the DuoLink in situ proximity ligation assay (PLA) (Olink Bioscience, Uppsala, Sweden) was used according to the manufacturer's protocol. Reactions were performed on sections (6-8 µm) of formalinfixed (4% in PBS, overnight at 4°C) and paraffin-embedded mouse testes. An antigen retrieval step in 0.01 M citrate buffer pH 6.0 was performed before the procedure. Sections were washed in PBS (3 × 5 min), incubated in Blocking Solution (Olink Bioscience) and immunolabeled (overnight, 4°C) with primary antibodies: rabbit anti-SPEN (N-terminal (1:200; PAB20849, Abnova), internal (1:170; A301-119A, Bethyl Laboratories) or C-terminal (1:30; NB100-58799, Novus Biologicals)), and goat anti-MTA1 (1:70; sc-9446, Santa Cruz Biotechnology) diluted in 1% BSA in PBS; negative controls were proceeded without one primary antibody or both. Then, the secondary antibodies with attached PLA probes (PLA Probe anti-Rabbit PLUS and PLA Probe anti-Goat MINUS; supplied in the DuoLink kit) were used. Signals of analyzed complexes were observed using Zeiss Axio Imager.M2 fluorescent microscope with AxioCam camera and AxioVision (Rel.4.8) imaging system; red fluorescence signal indicated a close proximity (<40 nm) of proteins recognized by both antibodies (Fredriksson et al. 2002) (link). Images were taken at ×1008 magnification in several focal planes.
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