Alexa flour 488 phalloidin

Untreated and MNPs@SiO₂(RITC)-treated cells were incubated on soft or rigid surfaces for 12 h and fixed with 4% paraformaldehyde in PBS for 15 min to study the spreading area, aspect ratio, and filopodia structure of the cells. They were then permeabilized with 0.1% Triton X-100 in PBS for 15 min. Next, the actin structures of the cells were labeled with Alexa Flour 488 phalloidin (Thermo Fisher Scientific) (1:2500) in PBS for 30 min at RT. The cell nuclei were then labeled with 0.2 mg/mL of 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) (1:10,000) in PBS for 15 min at RT. Finally, the stained cells were imaged using a DeltaVision microscope (GE Healthcare, Chicago, IL, USA) equipped with a CoolSnap HQ2 camera (Photometrics, Tucson, AZ, USA). The cell spreading area and cell aspect ratio were measured using FIJI ImageJ (NIH), and the filopodia of cells were analyzed using a FIJI ImageJ plug-in called FiloQuant [49 ].

Cells were fixed with 4% paraformaldehyde (PFA) (Polysciences, Warrington, FL, USA). To block unspecific binding sites, the fixed cells were incubated with blocking buffer containing 10% normal goat or donkey serum, 1% BSA, 0.5% Triton, and 0.05% Tween for 2 h at room temperature. Incubation of the primary antibody was performed at 4 °C overnight in staining buffer (blocking buffer diluted 1:1 with PBS). After at least 16 h of incubation, the cells were washed three times with PBS/0.05% Tween and incubated with a 1:500 dilution of secondary antibodies dilution. Afterwards, the cells were washed again three times with PBS/0.05% Tween, nuclei were stained with Hoechst 1:5000 (Thermo Fisher Scientific, Waltham, MA, USA), and podocyte cytoskeleton was stained with Alexa Flour 488 phalloidin (Thermo Fisher Scientific) (1:400). Images were captured using a fluorescence microscope (LSM700; Zeiss, Oberkochen, Germany) with Zenblue software (Zeiss). Individual channel images were processed and merged with Fiji. Detailed information on the used antibodies are given in Supplementary Table S3.

For immunocytochemistry and confocal laser scanning microscopy (CLSM) colony pieces were dissected into individual zooids and freed from the ectocyst, occasionally the entire cystid. To improve permeability, the specimens were treated with 0.1 mol L⁻¹ PB including 2% Triton‐X 100 and 2% dimethylsulphoxide (PBT) for 24 h. For Factin staining, Alexa flour 488 phalloidin (Cat# A12379; Thermo Fisher Scientific) was applied at a dilution 1:40 and nuclear‐counterstaining was done using 4′,6‐diamidino‐2‐phenylindole (Invitrogen) at a dilution approximately 1:300. After staining, the specimens were rinsed several times in 0.1 mol L⁻¹ PB and mounted on object slides in Flouromount G (Southern Biotech). Scans were carried out on a Leica TCS SP5 II CLSM (Leica Microsystems). Image processing and analysis were done using FIJI (Schindelin et al., 2012 (link)) and Amira software (v. 2022; Thermo Fischer Scientific). Individual muscles were segmented using the segmentation editor of Amira, volume renderings were produced using the volren and volume rendering module in combination with several orthoslices. Snapshots were exported to be further processed using Adobe Photoshop.

Cells were fixed with 4% paraformaldehyde (PFA) (Polysciences, Warrington, PA, USA). Unspecific binding sites of the fixed cells were blocked by incubation with blocking buffer containing 10% normal goat or donkey serum, 1% BSA, 0.5% Triton, and 0.05% Tween, for 2h at room temperature. The primary antibody was diluted 1:1 in blocking buffer with PBS and incubated at 4° C overnight (or at least 16h). After incubation the cells were washed three times with PBS/0.05% Tween and the secondary antibodies were diluted the same way as the primary antibodies with a 1:500 dilution. After 1h of secondary antibody incubation the cells were washed again three times with PBS/0.05% Tween and nuclei were stained with Hoechst 1:5000 (Thermo Fisher Scientific) and cytoskeleton was stained with Alexa Flour 488 phalloidin (Thermo Fisher Scientific) (1:400). Images were captured using a fluorescence microscope (LSM700; Zeiss, Oberkochen, Germany) with Zenblue software (Zeiss). Individual channel images were processed with Fiji. Detailed Information of the used antibodies are given in Supplementary Table 2.

All samples were fixed with 4 (wt. %/vol. %) paraformaldehyde in PBS for 30 min. Then, they were washed 2–3 times with PBS with a 5 min waiting time between each washing step. Then, the samples were placed in 1% Triton-X overnight and washed afterward with PBS again. All fixed samples were stained with 1 μM Alexa-Flour488 Phalloidin (Thermo-Fisher, Waltham) and 2 mg/ml Hoechst 34580 (Invitrogen, H21486). Since imaging through the collagen sheet is impossible with good quality, a circular piece (d = 8 mm) of the collagen network around the cell aggregate in question is punched out and then flipped upside down in a new well of a 24 μ-well plate. Through this approach, the spheroid directly faces the objective of the inverted microscope and can be easily imaged. However, the punch out process can lead to disturbances in the collagen matrix and tension relaxation¹³ (link) may occur, so that the collagen structure including alignment may not be representative of the situation during the vital measurements.

After treatments with aggregrated HiLyte Fluor-555-labeled Aβ₄₂ (Anaspec, Fremont, CA), astrocytes were washed with 3 times with PBS for 5 min, before fixation with 4% formaldehyde for 10 min at room temperature, and permeabilization with 0.1% TritonX-100 for 5 min. After careful washing, cells were incubated with 100 nM of Alexaflour 488 phalloidin (Thermofisher A12379) and Topro3 (Thermofisher T3605) diluted in 1% BSA/PBS for 10 min at room temperature. Cells were washed then mounted on coverslips with 90% glycerol, 20 mM Tris–HCL (1 M, pH 8.8), and 0.5% (w/v) p-Phenylenediamine (CSHB Protocol). Images were captured with either a Leica TCS-SP8-AOBS or Zeiss LSM 800 Confocal microscope.
Images were quantified with ImageJ software. Integrated density of HiLyte Fluor-555-labeled Aβ₄₂ fluorescence intensity with the bounds of a mask based upon phalloidin defined maximum threshold was measured and normalized to phalloidin mask area and nuclei marked by Topro3.