Ab235138

The podocytes were fixed by 4% paraformaldehyde, permeabilized by 0.1% TritonX-100 (T8787, Sigma) for 10 min on ice, and incubated with rhodamine-phalloidin (ab235138, Abcam) (1:100) for 1 h at room temperature. The cells were observed on a confocal microscope (20 x, LSM 780, Zeiss). ImageJ is used for measuring the mean fluorescent intensity (MFI) of whole field cells (20 × objective) from three independent assays.

To perform a morphological analysis of transfected HUVECs, staining with Rhodamine-phalloidin was conducted in order to visualize the actin cytoskeleton and filopodia. The HUVECs were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and then incubated with Rhodamine-conjugated phalloidin (Abcam, ab235138, 1:1000) for 30 min at room temperature. Subsequently, images were captured using an inverted microscope (Olympus Corp, IX73P1F) and the number and average length of filopodia per cell were determined using the FiloQuant plugin in Fiji (ImageJ).

Rat hippocampal neurons were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.25% Triton X-100 in phosphate buffered saline (PBS) for another 5 min. After washing, the cells were blocked with 5% bovine serum albumin (BSA) in PBS for 45 min. Then, the cells were incubated with diluted primary antibodies for two hours. Subsequently, samples were incubated with fluorescence-conjugated secondary antibodies for 45 min. We captured fluorescence images using Leica-LCS-SP8-STED confocal laser-scanning microscopes with 63× oil immersion objectives and an additional 2× zoom. Confocal image analysis and quantification were conducted as described before [40 (link)]. ImageJ was used for data analysis and quantification.
We transfected the ERVWE1, Wnt5a, or miR-141-3p inhibitor expression plasmid into rat hippocampal neurons at 5 days in vitro (DIV5). A sholl analysis was conducted on DIV14 and DIV21 to examine dendritic complexity and spine morphology of rat hippocampal neurons stained with microtubule associated protein 2 (MAP2, dilution 1:100, Abcam, ab5392, Cambridge, UK) and phalloidin (dilution 1:100, Abcam, ab235138, Cambridge, UK), respectively. Thus, we counted dendritic intersections across each 10 μm circle up to 150 μm distal to the soma.

For staining of cytoskeletal F-actin, 5 × 10⁴ cells were seeded on sterile coverslips into 24-well plates and cultured for 72 h. Cells were washed and fixed as described above. After blocking for 1 h at room temperature with 1% BSA/PBS, cells were stained with phalloidin (#ab235138, Abcam, 1:2000 in PBS (#D8537, Merck)) for 30 min at RT, and finally cover-slipped with Mounting Medium for Fluorescence with DAPI (#H-1200-10, Vector Laboratories). Slides were stored at −20 °C in the dark and imaged with a fluorescence microscope (Olympus, Tokyo, Japan; IXplore Live-System).

After shear stress stimulation, hPDLSCs were fixed with 4% (v/v) paraformaldehyde (BOSTER) in DI water and incubated with rhodamine–phalloidin (dilution 1:1000) (cat. No. ab235138, Abcam, Cambridge, UK). Stained cells were then counterstained with DAPI (dilution 1:2000) (TOCRIS bioscience, Bristol, UK). Then, 50% glycerol was added to the plate and stored at 4 °C. Apotome microscopy (Axio Observer Z1 and ZEN pro, ZEISS International, Oberkochen, Germany) was performed.

Briefly, cells were fixed in 4% formaldehyde in PBS 1X for 30 min at room temperature. Coverslips were incubated with 0.1% Triton X-100 for 3 min. Following washing with PBS 1X, cells were blocked for 40 min at room temperature with 0.2% BSA in PBS 1X, and incubated with Phalloidin 1X (ab235138, Abcam, Cambridge, UK) for 30 min at room temperature with anti-FAK antibodies overnight at 4 °C. Then, stained cells were washed with PBS 1X for 15 min and mounted for confocal microscopy.