Lsm 800 meta

The distribution of mitochondria and the ER in living oocytes was detected by Mito-Tracker Red CMRos (1:600) (Cat# M7512, Invitrogen, Eugene, OR, United States) or ER-Tracker Red (1:500) (C1041 Beyotime Biotechnology Shanghai, China) in an M2 medium at 37°C for 30 min. Then we washed the oocytes three times and scanned the live oocytes with a laser confocal microscope by Zeiss LSM 800 META.

RNA isolation and cDNA synthesis were carried out according to standard procedures [26 (link)]. PtFucTs sequences were fused to the upstream of eGFP and cloned into pPha-NR vector (GenBank: JN180663) via EasyGeno kit (Tiangen, Biotech Co., China). During the fusion, the stop codon of PtFucTs was deleted. The primers were designed via the EasyGeno Primer website (http://123.56.75.195/#enidx). pPha-NR-PtFucTs-eGFP plasmids were transfected into P. tricornutum cells on the 5th day of culture. Afterwards, the clones were analyzed with confocal laser scanning microscope (LSM 800 Meta, Carl Zeiss, Germany). An argon laser at 488 nm was used to activate the fluorescence of eGFP and plastid autofluorescence. While the fluorescence was detected at bandwidths of 500–520 nm (green fluorescence of eGFP), 580–600 nm (magenta fluorescence of mRFP) and 625–720 nm (red fluorescence of plastid), respectively [21 (link)]. All primer sequences used in this paper were shown in Additional file 2: Table S1.

Lysosome Red (1:10,000) (C1046 Beyotime Biotechnology Shanghai, China) was used to detect the distribution of lysosome in living oocytes, which were incubated with an M2 medium at 37°C for 30 min. The oocytes were washed three times and then examined by a confocal laser-scanning microscope (Zeiss LSM 800 META, Germany).

The embryos were immobilized with 4% (w/v) paraformaldehyde in PBS 1 h and then permeabilized with 1% Triton X-100 (in PBS) for 15 min at room temperature, where after blocked by blocking buffer (1% BSA-addition of PBS) 1 h at room temperature. For anti-α-tubulin-FITC and anti-gamaH2A.X staining, embryos were incubated with primary antibodies (anti-α-tubulin-FITC, 1:100; anti-gamnaH2A.X, 1:200) overnight. The embryos were further incubated with corresponding secondary antibodies (Alexa Fluor 594 bound goat anti-rabbit antibody, 1:200) for 1 h at room temperature. Then, the embryos were stained with Hoechst 33342 (10 mg/mL in PBS) for 15 min and samples were mounted on glass slides. Finally, the embryos were examined with a confocal laser-scanning microscope (Zeiss LSM 800 META, Germany). Fluorescence intensity was analyzed by image J software. To avoid errors, the embryos of the treatment and control groups were sealed on a sheet of glass and scanned with the same parameters to standardize different replicates. Average fluorescence intensity per unit area of the target region was calculated using image J. When the fluorescence intensity is counted, the embryos with extremely strong and weak fluorescence intensity are excluded. The average fluorescence intensity of all embryos was used as the final average fluorescence intensity.

Surface markers on hUCMSCs were analyzed by flow cytometry. After trypsinization, approximately 1 × 10⁶ cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Collected cells were then incubated with indicated PE-conjugated antibodies CD13, CD29, CD31, CD34, CD44, CD45, CD73, CD117, HLA-ABC, and HLA-DR (eBioscience, Shanghai, China) at room temperature for 2 h. Control samples were incubated with PE-conjugated mouse IgG1 isotype antibodies. After incubation, cells were washed with PBS and centrifuged to remove unbound antibodies. Cells were resuspended in 1 ml PBS and analyzed by flow cytometry using the Accuri C6 cytometer (BD, Franklin Lakes, NJ). Surface markers on PMVs were measured by fluorescence staining. PMVs from hUCMSCs were adhered to a 35-mm glass-bottom dish (In Vitro Scientific, Sunnyvale, CA), fixed with 4% paraformaldehyde, and incubated with the above PE-conjugated antibodies at room temperature for 2 h. After washing, PMVs were examined and photographed under a confocal microscope (LSM 800 Meta, Carl Zeiss, Germany).

4.5 × 10⁴ CT26 cells were seeded in µ‐Slide 8 well (Ibidi, USA) for 24 h and then treated with 100 × 10⁻⁹m of HP, FHP‐1, and FHP‐2. After 24 h, the cells were washed with PBS prior to incubation with 10 × 10⁻⁶m of DCF‐DA at 37 °C for 30 min. Following washing with PBS, the cells were stained with ER‐Tracker Red (500 × 10⁻⁹m, Invitrogen Co.) at 37 °C for 30 min. The stained cells were washed with PBS containing Pluronic F‐127 (0.1%) and visualized with confocal laser scanning microscopy (LSM 800 META, ZEISS, Germany).