Lsm 710 nlo laser scanning confocal microscope

Differentiated cells were fixed for 30 min with 4% paraformaldehyde, and blocked with 5% normal goat serum and 1% BSA in 0.2% Triton X-100 for 45 min. Primary antibodies were diluted in 5% normal goat serum and incubated with the samples overnight at 4°C. The appropriate fluorescently labeled secondary antibodies were applied for 2 h at room temperature. The nuclei were counter stained with 4, 6-diamidinodiamidino-2-phenylindole (DAPI, 10 mg/ml, Life Technologies). Negative control (omit primary antibody) was included in all immunofluorescent staining. Immuno labeled cells were viewed and counted using Zeiss LSM 710 NLO laser scanning confocal microscope (Jena, Germany). The percentage of MAP-2/TH/DAPI positive cells was calculated within 10 randomly selected visual fields. The following primary antibodies were used: 1:500 rabbit anti-TH (Millipore, AB5935), 1:500 mouse anti-MAP2 (Abcam, ab11267) 1:200 goat anti-GIRK2 (Abcam, ab65096). The secondary antibodies were as follows: Alexa Fluor 488 goat anti-mouse (1:400, ab150113, Abcam), Alexa Fluor 488 donkey anti-goat (1:400, ab150129, Abcam) and Alexa fluor 594 goat anti-rabbit (1:400, ab150080, Abcam).

MDA‐MB‐231 cells (1.5 × 10⁴) were seeded onto Matrigel‐coated glass coverslips and cultured for 24 h in DMEM supplemented with 10% FCS at 37 °C. After two washes with PBS, cells were cultured with or without κE (50 μg·mL⁻¹) for 6 or 24 h in DMEM without FCS, then fixed in 2% paraformaldehyde, washed with PBS and incubated for 1 h at room temperature in blocking solution (PBS containing 3% bovine serum albumin). Cells were then incubated overnight at 4 °C with MMP‐14 primary antibody (rabbit anti‐human MMP‐14 antibody, clone EP1264Y, GeneTex), washed and stained for 30 min at room temperature with goat anti‐rabbit secondary antibody (Invitrogen/Thermo Fisher Scientific) conjugated to Alexa Fluor 568 and Hoescht reagent. Finally, they were mounted in FluorSave Reagent (Merck, Darmstadt, Germany) and examined using an LSM710 NLO laser scanning confocal microscope (Carl Zeiss Axio Observer). Emitted fluorescence was detected through the use of the appropriate filter set, and representative images from three separate experiments were treated and merged with imagej software (NIH, Bethesda, MD, USA).

Full-length RGLG3 and RGLG4 were amplified by PCR, and each was cloned into pRTL-GFP, generating a construct expressing an N-terminal GFP fusion protein. Arabidopsis mesophyll protoplast preparation, transfection and 4,6-diamidino-2-phenylindole (DAPI, 1mg/ml; Sigma, USA) staining were performed as described (He et al., 2011b (link)). Confocal imaging was performed with a Zeiss LSM 710 NLO laser scanning confocal microscope (excitation 488nm; emission 505–550nm). Expression of GFP alone (from the control pRTL-GFP), GFP-RGLG3 and GFP-RGLG4 was confirmed by western blotting using anti-GFP (Abcam, Germany) as the primary antibody and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Promega, USA) as the secondary antibody.