Lsm710

To examine the transfection efficiency, flow cytometry and confocal assay were performed. GL261 cells were cultured until 80–90% confluent in 6-well plates. Then EGFP-Nano-reshaper loaded with plasmid (2 µg) were added in the presence of Opti-MEM. The medium was replaced with DMEM 6 h after the transfection and incubated for another 48 h. The non-EGFP coding vector plasmid-loaded nanoparticle (ApoE-pVector@CaCP) was set as the negative control (NC), and the commercial reagent, Hieff Trans^TM Liposomal Transfection Reagent was set as a positive control following the manufacturer’s protocol. After the imaging, the expression of EGFP was measured via flow cytometry and observed by confocal laser scanning microscopy (CLSM, LSM710, Leica, Germany).

Microscopic analyses were carried out using Zeiss LSM 510 Meta, a Zeiss LSM710, and Leica TCS SP5 laser scanning confocal microscopes. For mTurquoise2, an excitation wavelength of 458 nm was used and emission was collected between 460 and 520 nm. GFP was excited with a 488 nm laser line and fluorescence was collected between 490 and 530 nm. mVenus was excited with a 514 nm laser line and fluorescence was collected between 520 and 560 nm. For propidium iodide (PI) fluorescence, an excitation wavelength of 488 nm was used, whereas emission was collected between 600 and 670 nm. RFP and FM4-64 fluorescence was excited at 561 nm and emission was collected between 560 and 620 nm (RFP) or between 675 and 790 nm (FM4-64). SCRI Renaissance 2200 was excited with the 405 nm laser and fluorescence was collected between 425 and 475 nm. Images were analysed with ImageJ/Fiji (https://imagej.net/software/fiji/).

Whole-mount samples were stained and cleared with a modified 3DISCO protocol^{37 (link)}. In short, samples stored in PBS-GT were incubated with primary antibodies in PBS-GT with shaking, for 36 h at RT. Excessive antibody was removed by thorough washing in PBS-GT for 6–12 h and refreshing the solution every 1–2 h. Incubation with fluorophore-coupled secondary antibodies (Molecular Probes) in PBS-GT for 36 h was followed by thorough washing in PBS-GT as described above. When necessary, samples were dehydrated in an ascending Tetrahydrofuran (Sigma, #186562) series (50%, 70%, 3 × 100%; 60 min each), and subsequently cleared in dichloromethane (Sigma, #270997) for 30 min and eventually immersed in benzyl-ether (Sigma, #108014). Non-cleared samples were imaged in 35 mm glass-bottom dishes (Ibidi, #81218) using a laser scanning confocal microscope (Zeiss LSM710) or SP8 Multiphoton microscope (Leica). Cleared samples were imaged whilst submerged in benzyl-ether with a light-sheet fluorescence microscope (LaVision BioTec).

Intact guts from adult female flies were dissected in PBS, fixed in fixative solution (4% formaldehyde in a pH 7.5 solution containing 100 mM glutamic acid, 25 mM KCl, 20 mM MgSO₄, 4 mM sodium phosphate dibasic, 1 mM MgCl₂), washed twice in wash buffer (1× PBS, 0.5% bovine serum albumin and 0.1% Triton X-100) first for 10 min at room temperature (RT), and then for 1 h at 4 °C in a shaker. Primary and secondary antibodies were incubated overnight at 4 °C or for 4 h at RT. All antibodies were diluted in wash buffer. For the immunostaining of Delta antibody we used a methanol-heptane fixation method described in ref. ⁷² followed by the above described wash steps and antibodies incubations. Hoechst was used to stain DNA. All Images were taken on a Zeiss LSM 710 or a Leica SP5 confocal microscope and processed using Adobe Photoshop, Illustrator and FIJI is just Image J (FIJI).

Cells were fixed in 4% PBS (Fisher Scientific UK, 12579099)-paraformaldehyde for 15 min, incubated in 0.1% Triton-X-100 (Fisher Scientific UK, 11471632) for 5 min on ice, then in 0.2% fish skin gelatin (Sigma, G7041) in PBS for 1 h and stained for 1 h with an anti-E-cadherin (1:100, Santa Cruz sc-21791, mouse monoclonal 67A4) antibody. Protein expression was detected using Alexa Fluor 488 (1:400: Fisher Scientific UK) for 20 min. TO- PRO-3 (Invitrogen, T3605: 1:1000) was used to stain nucleic acids. For immunofluorescence staining of 3D cultures from MCF10A ER:HRAS V12 cells, acini were fixed with 4% paraformaldehyde for 40 min, permeabilized in 0.5% Triton X-100 for 10 min on ice and stained with Rhodamine-phalloidin (Molecular Probes, R415) for 1 h at room temperature. Acini were counterstained with DAPI. Samples were observed using a confocal microscope system (Carl Zeiss LSM 510 or LSM 710, or Leica SP8). Acquired images were analyzed using Photoshop (Adobe Systems, United States) according to the guidelines of the journal.

Time-lapse imaging was performed with a Zeiss LSM710 or Leica SP8 confocal microscope with a ×40, numerical aperture 1.3 inverted oil lens, a 488-nm argon laser, and a 568-nm green HeNe laser. The basal focal plane, which is about 1 μm beneath the basal surface, was selected during live imaging to maximize the basal Myo-II intensity. For the dynamics of ROCK–GFP, MBS–GFP, MBS-RFP, or Flw-YFP, the similar basal focal plane was selected to maximize the basal signal intensity, as basal MyoII dynamic imaging did. The same microscope setup was used when comparing intensity between different samples. To view the signals at different focal planes, images were taken at different Z-stack layers from the basal surface to the apical side.
FRET images of live cultured egg chambers were acquired with a Zeiss LSM710 microscope, by using a similar version of the protocol described in ref. ^{38 (link)}. A 458 nm laser was used to excite the sample. CFP and YFP emission signals were collected through channel I (470–510 nm) and channel II (525–600 nm), respectively. To capture single, high-resolution, and stationary images, a ×40/1.3 oil inverted objective was used. CFP and YFP images were acquired simultaneously for most of the experiments. Sequential acquisition of CFP and YFP channels in alternative orders were tested and gave the same result as simultaneous acquisition.