Perkin elmer tsa plus cyanine3 system

Immunofluorescence (IF) was conducted as described in detail previously (Jørgensen et al., 2018 (link)). In brief, sections (4 μm) were dewaxed and rehydrated using standard procedures, followed by heat-induced antigen retrieval (pressure cooker) in 0.01 M citrate buffer (pH 6) and subsequent peroxidase block in 3% (v/v) H₂O₂ in methanol for 30 min. Sections were blocked in normal chicken serum (NCS; Biosera, Ringmer, UK) diluted 1:5 in TBS containing 5% (w/v) BSA. The primary antibodies OCT4 (Santa Cruz, sc-5279) and FGF9 (Santa Cruz, sc-7876) were both diluted 1:100, while secondary peroxidase-conjugated chicken anti-mouse and chicken anti-rabbit, respectively, were diluted 1:200. Following incubation with the appropriate secondary antibody, slides were incubated with Tyr-Cy3 and Tyr-Fluorescein (Perkin Elmer-TSA-Plus Cyanine3 System; Perkin Elmer Life Sciences, Boston, MA, USA) according to the manufacturer’s instructions. Sections were counterstained with DAPI (Sigma) diluted 1:500 in TBS for 10 min. Finally, slides were mounted with Permafluor (Thermo Scientific, UK) and fluorescent images were captured using an Olympus BX61 microscope (Olympus).

Cell were cultured, fixed and pretreated according to the protocol for the RNAscope® Multiplex Fluorescent Reagent Kit v2 Assay provided by Advanced Cell Diagnostics (ACD, #323100, Nunc Lab Tek II 2 Well Glass Slides, #154461K). The probes were as follows: RNAscope® 3-plex Positive Control Probe (320861), RNAscope® Negative Control Probe – Bacillus subtilis dihydrodipicolinate reductase (dapB) gene (310043), RNAscope® Target Probe C1 (20ZZ probe named Hs-KRT80 targeting 294-1445 of NM_182507.2, 300031), RNAscope® Target Probe C2 (RNAscope® Probe - Hs-SQLE 465071, 300031), RNAscope® Target Probe C2 (20ZZ probe named Hs-SREBF1 targeting 958-2002 of NM_001005291.2, 300031) and RNAscope® Probe - Hs-HMGCR (RNAscope® Target Probes, 470561). The assay was run following the manufacturer’s instructions, hybridization was performed overnight. PerkinElmer TSA Plus Fluorophores (fluorescein, NEL741001KT and Cyanin 3, NEL744001KT) were diluted at 1:1300 and assigned to the channels HRP-C1 and HRP-C2, respectively. Fluorophores: PerkinElmer TSA Plus Fluorescein System (NEL741001KT) and PerkinElmer TSA Plus Cyanine 3 System (NEL744001KT). Samples were imaged using a ×60 objective with a Ti Nikon microscope equipped with a spinning disk (CAIRN) and analysed in Image J.