Invitrogen viewrna ish tissue 1 plex assay kit

RNA in situ hybridisation was conducted using Invitrogen ViewRNA ISH Tissue 1‐Plex Assay kits (Thermo Fisher Scientific) according to manufacturer's user guide and optimised for Medicago root and nodules sections (Kulikova et al., 2018). RNA ISH probe sets were designed and synthesised at Thermo Fisher Scientific. Catalogue numbers are VF1‐20312 for NIN (Medtr5g099060), VP2W7MP for CP2 (Medtr5g022560), VP7DPDU for SymCRK (Medtr3g079850), VPRWEMC for NAD1 (Medtr7g022640) and VF‐20311 for NF‐YA1 (Medtr1g056530). Each probe set was tested on tissue where it should not be present, in this case on noninoculated roots. As a negative control for each hybridisation procedure any probe set was omitted. In both cases no hybridisation signals were detected. The in situ images were taken with an AU5500B microscope equipped with a DFC425c camera (Leica).

RNA in situ hybridizations were performed as described by Liu et al., (2019 (link)) using the Invitrogen ViewRNA ISH Tissue1‐Plex Assay kits (Thermo Fisher) and in accordance with the manufacturer’s instructions. RNA in situ hybridization experiments were repeated three times. A detailed experimental procedure is provided in Methods S1.

Medicago roots hosting the AM fungus were fixed with 4% paraformaldehyde mixed with 3% glutaraldehyde in 50 mM phosphate buffer (pH 7.4) overnight, dehydrated in serial dilution of ethanol and then embedded in paraffin (Paraplast X‐tra; McCormick Scientific, St Louis, MO, USA) as described (Kulikova et al., 2018). Next, 7‐μm‐thick root sections were cut using an RJ2035 microtome (Leica). RNA in situ hybridisation was conducted using Invitrogen ViewRNA ISH Tissue 1‐Plex Assay kits (Thermo Fisher Scientific, Waltham, MA, USA) according to the user manual available online (shorturl.at/fuzPT).
RNA ISH probe sets were designed and synthesised by request at Thermo Fisher Scientific. A typical probe set consisted of c. 20 synthetic adjacent oligonucleotide pairs. Each of these pairs was composed of a 20‐bp primary sequence designed to target specific regions across the target mRNA sequence and a secondary extended sequence serving as a template for the amplification and detection of hybridisation signals. A probe set for RiNLE1 (catalogue number VF1‐6001202) covered the mRNA sequence from 2 bp to 613 bp and a probe set for MtPT4 (VF1‐19337) covered the region from 390 bp to 1296 bp. A probe set for MtPT4 was used as a positive control for in situ hybridisation. As a negative control, a RiNLE1 sense probe set (VPEPR3M) was used and probe sets were omitted for hybridisation.