High resolution laser confocal microscope

For ICC, cells grown in attached culture or tumour spheres were paraffin‐embedded, and the paraffin blocks were cut into slices. The slices of FFPE tissue have been deparaffinized, rehydrated and heated with citrate buffer (pH = 6.0) to retrieve antigen. After blocking antigens with normal goat serum for 2 h, the slices were added with anti‐DAB2IP polyclonal rabbit antibody (14 μg/ml; Sigma# SAB4301073) at 4°C overnight. Ten per cent Mayer's hematoxylin and the goat anti‐rabbit IgG secondary antibody (1:1000) were used to label DAB2IP and the cell nucleus, respectively. Images were captured through a microscope (Olympus BX51).
For immunofluorescence (IF), slices were deparaffinized, rehydrated, antigen‐retrieved and antigen‐blocked. Then, the antigens were labelled with anti β‐catenin rabbit antibody (1:100 dilution; CST # 8480) at 4°C overnight. A goat anti‐mouse/rabbit IgG secondary antibody (Alexa Fluor 488/594; Invitrogen) was used to label β‐catenin, while DAPI (Sigma‐Aldrich) was used to label the cell nuclei. Images were taken using a high‐resolution laser confocal microscope (Zeiss).

The vector was constructed using the method described by Jiang et al. (2017b) (link). The coding sequence of ZjSWEET2.2 was inserted downstream of the CaMV35S promoter in the plant expression vector pVBG23000-GFP, which includes the gene encoding green fluorescent protein (GFP). For the subcellular localization analysis, the positive vector CaMV35S-ZjSWEET2.2-GFP was transiently expressed in tobacco leaves after infiltration with Agrobacteriumtumefaciens strain GV3101 containing the vector. The pRT101-AtPIP2A-red fluorescent protein (RFP) was used as a plasma membrane marker, and was co-transformed with CaMV35S-ZjSWEET2.2-GFP. The tobacco plants were incubated at 25°C for 48 to 72 h, and the fluorescence of GFP was observed under a high resolution laser confocal microscope (Zeiss, Jena, Germany).
For the transient overexpression analysis, jujube leaves were vacuum-infiltrated with Agrobacterium containing the vector along with 500 µl Tween 20. The CaMV35S-GFP construct was used as a control. The leaves were cultured on Murashige and Skoog (MS) medium for 48 to 72 h in an incubator under a 12-h dark/12-h light photoperiod. The presence and relative amount of the transgene in transgenic leaves were determined by qRT-PCR.