Lms780

Adult rat CF and cardiomyocytes were plated onto glass chamber slides overnight and then incubated with α-BTX-FITC (1:2000, B13422, Thermo Fisher Scientific) for 2 hours in a 37°C humidified chamber. Cells were then fixed with 4% PFA, counterstained with Hoechst, and mounted and imaged on a confocal Zeiss LMS780 laser-scanning microscope.

ONS cells were cultured on 13 mm plastic coverslips (Sarstedt, Nümbrecht, Germany) with DMEM/F12 medium containing 10% FBS and 1% P/S. Cells were fixed either in 4% paraformaldehyde (PFA) or ice-cold methanol for 15 or 5 min and washed with PBS. PFA fixed samples were permeabilised with PBS containing 0.5% Triton-X 100. Blocking was performed at RT with 2% bovine serum album (BSA) (Sigma-Aldrich, St. Louis, MO, USA #A7906), 2% goat serum (Sigma-Aldrich, St. Louis, MO, USA #G9023) and 0.1% Triton-X 100 in PBS for 2 h. Primary antibodies for DCX (1:200, Abcam, #Ab52642), TUBB3 (1:500, Biolegend, San Diego, CA, USA #801202), Nestin (1:200, Abcam, Cambridge, UK #22035), AQP4 (1:200, Abcam, Cambridge, UK #9512 and S100b (1:200, Abcam, Cambridge, UK #Ab52642) were diluted in blocking solution and incubated overnight at 4 °C. The following day, cells were washed three times with 0.1% Triton-X 100 in PBS followed by incubation with secondary antibodies (Alexa Fluor −488, (#A-11029), −594 (#A-11037) and/or −647 (#A-21247), (all from Invitrogen, Waltham, MA, USA) for 2 h at RT in the dark and counterstained with a nuclear dye (Hoechst 1 µg/mL). Fluorescence images were captured using a confocal laser scanning microscope (LMS-780, Carl Zeiss AG, Oberkochen, Germany) and processed using Zeiss ZEN software version 1.1.2.0 (Carl Zeiss AG, Oberkochen, Germany).

Cells were plated at ~70% confluence on glass coverslips for 24 hours. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X (for pEGFR staining), and blocked in 5% goat serum for 30 minutes. After blocking, samples were incubated with anti-pEGFR antibody (1:100) or α7 nAChR (1:50), washed, and then incubated for 1 hour with appropriate fluorescent secondary antibody (1:200–1:300, Jackson ImmunoResearch), nuclear counterstained with Hoechst or DAPI, and mounted with Prolong Gold Antifade. Images were obtained with either a confocal Zeiss LMS780 laser-scanning microscope (×20 or ×40 magnification) of Nikon Eclipse E400 IF microscope. Confocal images were analyzed using Zen Blue (Zeiss) and ImageJ software (NIH). For each cell surface marker, slides were imaged at identical wide-field (magnification, scan speed, laser power, detector gain, and pinhole diameter) settings. The intensity of pEGFR was measured outlining the cells. Some images were then analyzed on NS Elements (Nikon) by measuring intensity of FITC channel divided over the measured area. Three to 5 images per animals were used and averaged together as 1.

Cells grown on coverslips were prepared as previously described (19 (link)). After incubation with the FLAG and the secondary anti-mouse Alexa-488 antibodies, images were obtained with an LMS 780 spectral confocal system (Zeiss, Jena, Germany).

WRL-68, MCF-7, E0771, Caco-2, HUVEC, and HBL-100 cells were seeded separately into 12-well plates containing complete culture medium at a density of approximately 1 × 10⁵ cells per well. Subsequently, the plates were incubated for the specified nanoparticles at 37 °C and 5% CO₂ in a CO₂ incubator. Upon completion of the incubation period, cells were rinsed thrice with PBS, fixed with 4% paraformaldehyde, and subjected to DAPI staining for visualization of the cellular nuclei. The acquired cellular images will undergo further analysis employing a confocal laser scanning microscope (Zeiss LMS-780, Thornwood, New York). For the evaluation of liver targeting efficacy, an excess of free GA was introduced half an hour prior to the experiment, thereby inducing ASGPR saturation.

Laser scanning confocal microscopy (Zeiss LMS 780, Germany) equipped with a digital camera was used to capture micrographs required for quantifications. For analyzing the regional distribution of cells in the entire CC, tile scanning was performed using up to four different fluorophores with minimal overlap, for immunostaining. To detect more than four fluorophores or to distinguish overlapping fluorophores, the emission fingerprinting technique was applied. Spectral reflectance imaging was conducted on non-stained sections to visualize myelin in the CC. These images were then imported to ImageJ software (NIH) to quantify different cell populations, fluorescent intensity of myelin-specific proteins, or the total amount of myelin present in the CC. Nine sections were examined for each animal at each of the rostral, middle, and caudal segments of the CC, between levels 1.10 and 1.94 mm relative to Bregma. In all three segments, we quantified cell density in the mediolateral axis of the corpus callosum relative to the distance from the dorsolateral corner of the V-SVZ. To determine the amount of myelin-specific proteins or the total amount of myelin, we measured the mean gray value using ImageJ software (NIH) and reported it as optical density (OD). The quantitative data is presented as mean ± SEM. All cell counts and measurements were performed in a blinded manner.

The PC12 cells were seeded in 6-well glass slides at a density of 2 × 10⁵ per well. After attachment, the cells were treated with PhGs (0.1 and 10 μg/mL) for 24 h and incubated with H₂O₂ for another 2 h after the PhGs were removed. After incubation, the cells were washed in cold PBS and fixed in 4% paraformaldehyde for 15 min at room temperature, followed by membrane permeabilization using 0.5% Triton X-100 in PBS for 5 min. The cells were incubated with anti-Nrf2 Rabbit IgG (Abcam) with 5% FBS overnight at 4 °C. Then, the secondary anti-rabbit antibodies conjugated with FITC were applied to the cells for 20 min at room temperature. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) at the final preparation step. The slides were rinsed briefly with PBS, air-dried, and mounted in an anti-fluorescence in fading medium. The slides were visualized under a laser confocal microscope (LMS780, Zeiss, Germany).