Activation of TLR signaling was evaluated utilizing a proximity ligand assay as previously described (37 ). Briefly, CLL LN cells were applied to a ibidi μ-slide with 15 wells (Sigma) and allowed to adhere for 30 minutes. The cells were fixed with 4% PFA for 10 minutes, permeabilized with 90% methanol for 20 minutes and blocked with Duolink block solution for one hour. Primary antibodies were prepared in Duolink antibody dilution buffer and incubated overnight 4C (1:200 dilution; Sigma). Cells were counterstained with CD20 488nm (eBioscience). Primary antibodies included, TLR9 (rabbit), IRAK1 (rabbit) and pIκBα (mouse) from Cell Signaling and MYD88 (rabbit) from Abcam. The cells were incubated with secondary antibodies (Duolink PLUS and MINUS; Sigma) at 37C for one hour, followed by a ligation mixture (Sigma) at 37C for 30 minutes and an amplification mixture (Sigma) at 37 for 100 minutes, with washes in between. The wells were mounted with Prolong Gold mounting medium with DAPI and visualized on a Zeiss LSM 880 NL Airyscan microscope (Zeiss). Positive and negative controls for the assay were previously published (34 (link)).
Amplification mixture
Manufactured by Merck Group
Amplification mixture is a laboratory reagent used in molecular biology techniques, such as Polymerase Chain Reaction (PCR), to facilitate the amplification of target DNA or RNA sequences. The mixture contains the necessary components, including a thermostable DNA polymerase enzyme, nucleotides, buffers, and other essential elements, to enable the exponential replication of specific genetic material.
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Lab products found in correlation
2 protocols using amplification mixture
1
Proximity Ligation Assay for TLR Activation
2
Proximity Ligation Assay for Studying EGFR Interactions
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Proximity ligation assays were performed by Duolink In Situ-Fluorescence Detection Reagent Red (Sigma-Aldrich) according to the manufacturer’s instructions. In brief, cells were seeded on coverslips and cultured overnight followed by transfection of Myc-EGFR expression plasmid for 48 h. Cells were then fixed, permeabilized, and incubated with the primary antibody overnight at 4°C. On the following day, the cells were washed with PBS and incubated with PLA probes for 1 h at 37°C. The cells were then washed twice with buffer A (Sigma-Aldrich), incubated with ligation mixture for 1 h at 37°C, washed twice with buffer A, and incubated in amplification mixture (Sigma-Aldrich) for 100 min at 37°C. After three washes with buffer B (Sigma-Aldrich), the coverslips were mounted with DAPI Fluoromount-G (SouthernBiotech). Images were captured using a Zeiss LSM 700 Confocal microscope (Zeiss, Oberkochen, Germany).
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