Fluorescence in situ hybridization of Arabidopsis thaliana protoplasts (FISH) was performed following an established protocol39 (link). Briefly, mock and TCV-infected protoplasts were fixed with methanol and hybridized with 4 μM Cy3-labelled probe complementary to bases 1210–1259 of the TCV genome (5′-Cy3-GCCTTCACGAATGTTTTGAGTTCTGCGTCCTTCCGGGATACCGGTCTGTC-3′). Cells were collected and washed with 2X SSC + 10% formamide before mounting with ProLong Gold + DAPI (Invitrogen). Cells were imaged using a laser scanning LSM 710 AxioObserver confocal microscope (Carl Zeiss). Images were acquired using a 63x/1.40 Oil DIC objective with 405 nm and 561 nm filters for DAPI and Cy3 respectively.
Zebrafish larvae were infected as described above and fixed at 24 hrs post-infection. Whole-mount FISH was performed using a FITC-labeled SINV capsid probe amplified with anti-FITC-AF488. Hoechst dye was used for detection of nuclei. Single confocal plains from an infected region in the tail were imaged using a 40x objective.
Zebrafish larvae were infected as described above and fixed at 24 hrs post-infection. Whole-mount FISH was performed using a FITC-labeled SINV capsid probe amplified with anti-FITC-AF488. Hoechst dye was used for detection of nuclei. Single confocal plains from an infected region in the tail were imaged using a 40x objective.
