Hc pl apo cs2

The cochleae were gently freed from the capsule, and the staining and clearing were performed according to an earlier study (Hirashima and Adachi, 2015 (link)). Briefly, the samples were fixed with 4% PFA in PBS overnight at 4°C and then blocked by incubation in 10% normal goat serum (Abcam, #ab156046) diluted in 0.1% Triton X-100/PBS (PBT) for 3 hr at 37°C. The samples were treated with primary antibodies overnight at 4°C, washed in 0.1% PBT, and subsequently treated with secondary antibodies conjugated to either Alexa Fluor 546 or Alexa Fluor 647 overnight at 4°C. For counter staining of nucleus, we used DAPI (Dojindo Molecular Technologies, #D523-10, 1:200 dilution). The samples were mounted with 10 µL of 1% agarose gel onto a glass-based dish (Greiner Bio-One, #627871) for stable imaging. Then, the samples were immersed with the CUBIC-R+ (Tokyo Chemical Industry Co., #T3741) solution for optical clearing. Images were acquired using the confocal laser scanning platform Leica TCS SP8 equipped with the hybrid detector Leica HyD with the ×40 objective lens (NA = 1.3, WD = 240 μm, HC PL APO CS2, Leica) and the ×20 objective lens (NA = 0.75, WD = 680 µm, HC PL APO CS2, Leica) and the Olympus FluoView FV1000 with the ×30 objective lens (NA = 1.05, WD = 0.8 mm, UPLSAPO30XS, Olympus).

Z-stack images (512 × 512 pixel; 1 μm) were acquired using an 8-year-old confocal microscope SP8 (Leica Microsystems) equipped with an HC PL APO CS2 with 40× numerical aperture 1.3 oil objective, an ultraviolet diode (405 nm), and four lasers in visible range wavelengths (405, 488, 532, 552, and 635 nm). The setup was made up of five detectors [three hybrid detectors with high quantum yield compared to classical photomultiplier (PMT) detectors and two PMTs]. Mosaic sequential images were acquired using the between-stack configuration to simultaneously collect individual seven or eight channels and tiles before merging them to obtain one single image. Use of the between-stack configuration and the modulation of the detectors’ detection windows help reduce the leaking of fluorophores. Last, a digital zoom of 1.9 was applied during the acquisition and a mosaic multicolor image was obtained and exported into a .lif format. Detection windows and microscope configuration used in our study are listed in table S2.

The ability of dendrimer/drug complexes to induce the apoptosis and necrosis in MCF7 and HEP G2 cells was evaluated by confocal microscopy using double fluorescent dye staining of orange acridine (OA) and ethidium bromide (EB). Both of these fluorescent dyes can stain cell nuclei after intercalation with DNA. While OA can be easily uptaken by cells and stains the nucleus green, EB is able to stain the nucleus of damaged cells only in red. Confocal analysis allows the indication of the fractions of the cells that are early and late apoptotic, necrotic or healthy. The cells were treated with dendriplexes for 24 h and stained with dual fluorescent staining solution (2 μL) containing 100 μg/mL AO and 100 μg/mL EB for 2 min and covered with a coverslip. Then, cells were washed with PBS and visualized using a Leica TCS SP8 confocal microscope (Wetzlar, Germany) with the objective 63×/1.40 (HC PL APO CS2, Leica Microsystems, Wetzlar, Germany).
The cells with normal morphology and a green nucleus were recognized as living cells, with green nucleus and condensed or fragmented chromatin as early apoptotic cells, with condensed or fragmented red chromatin as late apoptotic cells and with red nucleus as necrotic cells. Leica Application Suite X (LAS X, Leica Microsystems, Germany) software was used for the export of images.

The nuclei isolated from the plants expressing pCRWN1::CRWN1-EYFP, pCRWN2::CRWN2-EYFP, pCRWN3::CRWN3-sGFP, and p35S::sGFP were placed on a cover slip and immunostained using anti-GFP antibody (ab290; Abcam) at 1:3000 and Alexa Fluor 488-conjugated anti-rabbit antibody at 1:1000, as mentioned above. The nuclei were mounted with ProLong™ Glass Antifade Mountant (P36982; Thermo Fisher Scientific) and stored at 4 °C. Super-resolution images were obtained using a STED microscope (SP8-gSTED; Leica Microsystems, Mannheim, Germany) equipped with a white light laser, a 592 nm STED laser, and a 100 × 1.40 N.A. oil immersion objective (HC PL APO CS2). The excitation wavelength at 488 nm and fluorescent signal in the range of 495–585 nm were detected by a HyD detector with 1.5–9.0-ns time gating. Images were obtained with 4 × line averaging and 2 × frame accumulation. The pixel size was set to 14 nm per pixel. Image processing was performed by deconvolution software (Huygens Professional ver.18.10.0p8 64b; Scientific Volume Imaging) with the default conditions. Images were further processed using ImageJ 1.51 g.

Confocal images were collected on a Leica SP8 confocal microscope (Leica Microsystems CMS GmbH, Wetzlar/Germany) equipped with an inverted microscope fitted with a 40× HC PL APO CS2 (1.10 NA) water immersion objective. Excitation of 6-AF and Nile Red was from the 488 nm and the 552 nm laser line of an OPS laser, respectively. The 1024 × 1024 images were collected using Leica Application Suite X software (Leica Microsystems CMS GmbH, Wetzlar/Germany).

The externalisation of phosphatidylserine as the first symptom of apoptosis was determined using an Annexin V-FITC staining kit (Biovision, USA). In parallel, propidium iodide (PI) staining was conducted for cellular membrane integrity analysis. After 48 and 72 h continuous incubation with PTX alone or PTX-NCs, at doses of 12.5, 25, and 50 nM, the cells were collected by trypsinization centrifuged at 1000 rpm for 10 min, stained with the supplied fluorochromes and analysed using a LSRII BD cytometer equipped with the FACS Diva LSR II software (BD Dickinson, New York, NY, USA), the obtained data are expressed as cell percentages [30 (link)]. In parallel, the molecular features of apoptosis, after staining the cells with Annexin V and Hoechst 33,258, were visualised with a confocal microscope (Leica SP-8, Leica Microsystems, Wetzlar, Germany) with a 63×/1.40 objective (HC PL APO CS2, Leica Microsystems). Leica Application Suite X software (LAS X, Leica Microsystems, Germany) was used to obtain the images.

CTL were seeded on poly-l-lysin-coated high-performance coverslips and fixed in 3% PFA (10 min, 37°C). Permeabilization and staining were performed in PBS 3% BSA, 0.1% saponin (Sigma) for 5 min and 60 min, respectively. Cells were stained with an anti-human Granzyme B antibody (10 µg/ml, clone GB11, Thermo Scientific) followed by a goat anti-mouse IgG Abberior Star 580 (Abberior Instruments). Coverslips (high-performance D = 0.17 mm ± 0.005, ZEISS, Germany) were mounted on microscopy slides using Mowiol-DABCO.
STED images were acquired with a Leica SP8 STED 3× microscope (Leica Microsystems, Germany) using a HC PL APO CS2 100×/1.4 oil immersion objective. To optimize resolution without bleaching in 3D, the 775 nm STED laser line was applied at the lowest power that can provide sufficient improvement in resolution compared to confocal. Z-stack series were acquired sequentially with the pulsed 532 nm laser. For image acquisition, we used the following parameters: three time average/line, 400 Hz scan speed. STED images were subsequently deconvoluted with Huygens Professional (SVI, USA) using the CMLE algorithm, with a signal to noise ratio (SNR) of 7. 3D image visualization was performed using the Fiji software.