F actin

Fresh cadaver dissections (not embalmed) were performed under institutional guidelines. The IRB committee reviewed this work and agreed that it does not require IRB approval or oversight. Seven (N = 7) dissections were performed in 5 male and 2 female cadavers. Ages ranged from 40–92 years (mean 82.5). The sagittal sinus was dissected to identify the bilateral CSF canalicular system, and photographs were obtained in selected specimens (Canon EOS 6D 50mm macro lens, Canon USA, Melville, NY). Fluoroscopy was performed after the CSF canalicular system was intubated with 0.25 mm silastic tubing (WPI, Sarasota, Florida) and injected with 0.2 mls of Omnipaque^TM (iohexol, GE Healthcare, Chicago, Illinois). The CSF channels in the sagittal sinus were injected in 1 specimen using 1–5μ fluorescent polymer spheres (Cospheric^TM, Santa Barbara, CA) after intubation with 22/24 gauge IV catheter (Baxter Healthcare Corporation, Deerfield, Illinois) and photographed under ambient/UV light. Neck dissections confirmed the anatomy of the CSF system in the neck in each specimen. One cervical biopsy was submitted for immunohistochemistry by 2-step indirect immunohistochemistry (IHC) for LYVE-1, D2-40, CD105, and F-actin (Sigma Aldrich, St. Louis, Missouri), and by direct IHC for the type-3 neurofilament protein vimentin (vimentin cy-3; Sigma Aldrich, St. Louis, Missouri) [15 (link)].

Chondrocytes were allowed to seed onto patterned equidistant micropillar substrates with different stiffnesses (total 2000 ​cells at 0.5 ​× ​0.5 ​cm substrate areas). To directly observe chondrocyte morphological changes, we did not starve cells. After seeding for 72 ​h, chondrocyte samples were collected for immunofluorescence. The immunofluorescent protocol was previously described [34 ]. In short, chondrocytes were fixed with paraformaldehyde (PFA, 4%, w/v) for 5–10 ​min, and then permeated with Triton X-100 (0.5%, v/v) for 5 ​min. The cells were required to wash three times in 1 ​× ​PBS before milk blockage in 5% BSA for 2 ​h. Then, the cells were washed and incubated with the primary antibody (1:200 dilution) at 4 ​°C overnight. Then the cells were added with the secondary antibody (AlexaFluor647, 1:400 dilution) for 2 ​h at RT. Finally, the cells were counterstained with F-actin (phalloidin, FITC-labeled) and Dapi (sigma). The immunofluorescent images were obtained through CLSM (Olympus, FV3000, Japan).

Larval and prepupal leg discs were fixed and stained following standard procedures. As primary antibodies we used rabbit and mouse anti-βGal, rabbit anti-Dys (a gift from L. Jiang and S.T. Crews), rabbit anti-DCas-3 (cleaved Drosophila Dcp-1, Cell Signaling Technology), rabbit anti-P-Mad (kindly provided by G. Morata). Mouse anti-Dl, anti-Dlg and anti-Tgo are from Developmental Studies Hybridoma Bank, University of Iowa. TRITC-phalloidin and Phalloidin-Atto 647N were used to stain F-actin (Sigma Aldrich), and secondary antibodies were coupled to Red-X, FITC and Cy5 fluorocromes (Alexa Fluor Dyes, Invitrogen).
To determine the levels of cell death in E(spl)-mβ and “fold” domains, we have performed Z-stack imaging of wild type (n = 8 prepupae leg discs) and dys²/dys³ mutants (n = 10 prepupae leg discs) and counted the number of D-Cas3 positive cells on each domain with the aid of the Fiji software. We selected for this analysis the joints between tarsal segments 2/3 and 3/4.

Human cardiac fibroblasts were seeded in chamber slides and grown to confluence with supplemented Iscove’s basal medium (Biochrom AG, Berlin, Germany) as described above. Cells were stimulated either with TNF-α or TGF-β for 6 h and fixed with 4% paraformaldehyde for subsequent fluorescence staining. Antibodies directed against NF-κB p65 ((D14E12) XP^® rabbit mAb #8242) or Smad2/3 ((D7G7) XP^® rabbit mAb #8685) (Cell Signaling, Frankfurt, Germany) were used. For visualization, Alexa Fluor 594 conjugated goat anti-rabbit IgG (H+L) was applied as a secondary antibody (R37117, Thermo Fisher Scientific, Schwerte, Germany). To visualize the cell shape, Alexa Fluor^® 488 Phalloidin (A12379, Thermo Fisher Scientific) was used to stain F-Actin and Hoechst 33,342 (B2261, Sigma-Aldrich, Taufkirchen, Germany) was used to stain nuclei. Documentation of the staining was done with an Axiovert M200 microscope equipped with an ApoTome modul (Zeiss, Jena, Germany).

In order to assess cellular morphology, immediately after functional testing engineered muscles from normoxic and hypoxic conditions were fixed by the drop wise addition of ice‐cold methanol‐acetone solution. Subsequently constructs were removed from their sutures and adhered to poly‐L‐lysine coated microscope slides and ringed with PAP pen (Sigma–Aldrich) before being permeablized with 1× Tris buffered saline (TBS: 0.5M) and 0.2% Triton x‐100 (Fisher Scientific, UK) for 2 h. Constructs were then incubated in rhodamine conjugated phalloidin (Fisher Scientific) diluted 1:200 in TBS to label F‐actin, and DAPI (Sigma–Aldrich) diluted 1:1000 in order to label cellular nuclei, and were incubated at room temperature in the dark for 3 h. Following 4 washes in distilled water, constructs were mounted on to glass coverslips using a drop of Fluoromount™ medium (Sigma–Aldrich). Images were captured using a Leica DM2500 Fluorescent microscope at 40 × magnifications and analysis was conducted using Image J software (NIH, USA), with a minimum of five images and 40 myotubes analyzed per engineered muscle.

Cells were fixed with 4% (w/v) paraformaldehyde for 15 min and permeabilized with 0.1% (v/v) Triton X-100 in PBS for 2 min, both at room temperature. Blocking was carried out for 1 hour with 10% (v/v) horse serum in PBS. Primary antibodies were allowed to bind for 1.5 hours and secondary antibodies for 25 min in blocking medium at room temperature. For detection, secondary antibodies (Invitrogen and Dianova) were coupled with Alexa488 (1∶400), Cy3 (1∶1000) or Cy5 (1∶100). To stain for filamentous actin (F-actin), phalloidin-TRITC (1∶1000, Sigma) was added during the secondary antibody incubation step. Nuclei were stained with DAPI or Hoechst 33258 (Sigma) for 2 min. Mounting of the cells was carried out using Mowiol.
Images were acquired using a Leica DM 6000 B microscope with a 40x/0.7NA objective lens or a 63x/1.32NA oil objective lens connected to a digital camera (DFC 360) and using LASAF software. Images were adjusted utilizing Adobe Photoshop.

Cells were seeded in duplicates into µ-Dish 35 mm (60 000 cells/ibidi dish). After 48 h, cells were washed with sterile phosphate-buffered saline (PBS, Thermo Fisher) before being fixed in 4% paraformaldehyde for 30 min (10 min at 4°C followed by 20 min at room temperature). Cells were washed and then permeabilized for 4 min in 0.2% Triton in PBS. Cells were washed again and incubated in blocking buffer (1% bovine serum albumin, and 2% normal goat serum, in PBS) for 1 h at room temperature before incubating with the primary antibody for 2 h. The following primary antibodies were used: Nestin (Abcam, ab22035, 1:100), Vimentin (Abcam, ab8069, 1:200), NG2 (Abcam, ab83178, 1:200), Ki67 (Abcam, ab16667, 1:200), Vinculin (FAK100, Sigma 1:200), and F-actin (FAK100, Sigma 1:500). Secondary antibodies were applied for 1 h (goat anti-mouse Alexa Fluor 488 preadsorbed, Abcam, 1:750 dilution and goat anti-rabbit Alex Fluor 594 preadsorbed, Abcam, 1:750). Nuclei were stained with DAPI (Roche).