Fv3000 laser confocal microscope

Cellular morphology was visualized after the 24 h culture using fluorescence microscopy. Briefly, the cell-laden constructs were fixed with 4% paraformaldehyde in PBS for 10 min at RT. After rinsing with PBS three times, the samples were placed in a permeabilization solution with 0.2% Triton X-100 for 10 min and rinsed again three times with fresh PBS. The cell-laden constructs were then blocked with 1% BSA in PBS for 1 h at RT. Immunostaining was performed with Alexa Fluor 488 Phalloidin (Gibco, Thermo Fisher, Waltham, MA, USA) and DAPI (Sigma-Aldrich, Missouri, USA) to visualize the F-actin and nuclei, respectively. The cell-laden constructs were visualized using an Olympus FV3000 laser confocal microscope.

The target fragments were amplified by using fresh R. palmatum cDNA as template and 1305-RpMYBs-F/R as primers. The target gene and pCAMBIA1305-GFP linearized vector were digested by QuickCut restriction enzyme (1305-RpMYB81 using the restriction endonucleases Xba I and BamH I; 1305-RpMYB98 using the restriction endonucleases Spe I and BamH I), and the gel was recovered to purify the product. T4 DNA ligase was used to link the vectors. The ligation product was transferred into E. coli DH5ɑ competent cells. Three positive colonies with target products were randomly selected. The cloned plasmid was sent to Beijing Aoke for sequencing.
The correct recombinant vector was transferred into Agrobacterium EHA105 competent cells (Shanghai Weidi) by the CaCl₂ method. The plasmids pCAMBIA1305-GFP, pCAMBIA1305-RpMYB81-GFP, and pCAMBIA1305-RpMYB98-GFP were transformed into the inner epidermis of the second to fourth leaves of N. benthamiana by Agrobacterium injection. After co-culture at 25 °C for 36 h, the cells were stained with 10 ug/mL 4',6-diamidino-2-phenylindole (DAPI) for 20 min, and then washed three times with pH 7.2 phosphate buffer. The slides were observed by a Olympus FV3000 laser confocal microscope (Olympus Company, Japan).

At 6 mah, the ovaries of gsdf^+/+ and gsdf^-/- XX fish were dissected, and the gonads were fixed in Bouin’s solution for 24 hours at room temperature, dehydrated, and embedded in paraffin. Tissue blocks were sectioned at 5 μm thickness, and immunohistochemistry was performed using the Gsdf, Amh and Cyp19a1a antibodies diluted at 1:1000, 1:500 and 1:2000, respectively. Gsdf and Amh antibodies were obtained from previous studies (7 (link), 26 (link)), where their specificities were checked. The Cyp19a1a antibody was provided by Professor Yoshitaka Nagahama, National Institute for Basic Biology Okazaki, Japan (39 (link)).
For IF, Alexa Fluor 586-conjugated secondary antibodies (Invitrogen, MA, USA) were diluted at 1:500 in blocking solution and incubated with tissue to detect primary antibodies. The nuclei were stained by 4’,6’-diamidine-2-phenylindole-dihydrochloride (DAPI) (Invitrogen, MA, USA). Immunohistochemical analysis was performed as described previously. Photographs were taken under an FV3000 Laser confocal microscope (Olympus, Tokyo, Japan). Finally, the positive signals were quantified using Image J software (National Institute of Health, MD, USA) following the instructions provided.

The paraffin sections of kidney tissues were dewaxed with water and then antigenically repaired using antigen repair solution (pH 8.0). The sections were shaken dry and then blocked with serum. They were incubated overnight at 4 °C with podocin rabbit polyclonal antibody (Proteintech, Wuhan, China) and cd2ap rabbit polyclonal antibody (Sigma, NY, USA), followed by secondary antibody, stained with 4′,6-diamidino-2-phenylindole (DAPI), and finally blocked and photographed under an Olympus FV3000 laser confocal microscope (Olympus, Japan).