Opal650 conjugated tyramide

In situ RNA hybridization was performed using the RNAscope Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics) according to manufacturer’s instructions. The following target probes were used: Mm-Dpt (Cat. #561511-C3), Mm-Tnfsf13b (Cat. #414891), Mm-Notch3 (Cat. #425171), Mm-Cxcl13 (Cat. #406311-C2). In brief, spleens were fixed in 10% formalin for 24 h at RT and embedded in paraffin. 3 μm-thick sections were baked in an oven at 60 °C for 1 h, then deparaffinized and dehydrated. Following rehydration, endogenous peroxidase activity was quenched with hydrogen peroxide for 10 min at RT. Target retrieval was carried out in a steamer (Braun, Type 3216) for 15 min at >98 °C. Protease treatment was performed with Protease Plus (Advanced Cell Diagnostics) for 20 min at 40 °C. Target probes were allowed to hybridize for 2 h at 40 °C, followed by signal amplification according to the ACD protocol. After the final amplification step, signal was detected with Opal520, Opal570 or Opal650-conjugated tyramide (Perkin Elmer). Sections incubated with a negative control probe (DapB) were analysed in parallel and a mix of positive control probes (Polr2a, Ppib, Ubc) was utilized to confirm RNA integrity. Images were acquired with an Olympus VS120 slide scanner fluorescence microscope using the VS-ASW-FL software (Olympus).

Spleens were fixed in 10% formalin for 24 h at room temperature and embedded in paraffin. In situ RNA hybridization was performed using the RNAscope Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics) with the following target probes: Mm-Oas3 (catalog no. 1054261-C2), Mm-Ifit3 (catalog no. 508251-C2), Mm-Cd3e (catalog no. 314721-C3), using a previously described protocol^{84 (link)}. After the final amplification step, hybridized probes were visualized using Cy3 or Opal650 conjugated tyramide (Perkin Elmer). Sections incubated with a negative control probe targeting the DapB gene from Bacillus subtilis were analyzed in parallel. Positive control probes against murine Ppib and Ubc were used to confirm RNA integrity in each detection channel for each of the analyzed spleens. Images were acquired with a NIKON Eclipse Ti2-E/Yokogawa CSU-W1 confocal spinning disk microscope with a CFI PlanApo λ ×20 objective/0.75 numerical aperture/1 mm working distance and a 50-µm pinhole disc.