Confocal sp8

All images for fixed samples apart from the whole-body imaging for IACS-010759 treated larvae, were acquired with Leica confocal SP8, using HCX PL APO 40x water immersion objective lens. Live imaging for MitoTracker were carried out using HCX PL APO 63x glycerol immersion objective lens. Appropriate lasers and collection gates were selected according to excitation and emission wavelength for each fluorophore. IACS-010759 treated larvae were imaged using Opera Phenix Plus (Revvity) with 20x water objective (NA 1.0). High throughput was achieved using ZF plates (Funakoshi).

Imaging was
performed using the Leica Confocal SP8× (2D cultures; 63×
magnifications) and the Leica Thunder microscope (10, 20, and 40×
magnifications for 3D GelMa constructs). Images were analyzed using
ImageJ software (Version 1.47). 3D images were composed in LASX (Version
3.5.7.23225).

HEK293 cells were cultured as described above and seeded on coverslips in 6-well plates. Cells were co-transfected with plasmids of human a4 N-terminal domain (WT and mutants) and fluorescent proteins using PolyJet Reagent (SignaGen) in accordance with the procedure recommended by the manufacturer. Cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.2% Triton-X and stained with anti-Flag antibody (Abcam, Cambridge, UK). For ionomycin treatment, cells were treated with 5 µM of ionomycin at room temperature for 15 min right before fixing. Images were acquired with a confocal microscope (Leica Confocal SP8, Wetzlar, Germany) using the 63× oil objective. Colocalization were measured with Mander’s coefficient M1, which calculates the percentage of total signal from the green channel which overlaps with the signal from the red channel [53 (link)].

Sections were produced and treated as described in the immuonohystochemistry section with the modification that after the ON incubation with primary antibodies, sections were incubated with a fluorescently labeled secondary antibody for 2 h at RT, and mounted using fluoromount-G.
Images were captured on a Nikon Ds-Qi1Mc digital camera attached to a Nikon Eclipse Ni microscope (6-µm sections), a Leica Confocal SP8 for colocalization studies (primary neurons and 40-µm sections), a PerkinElmer Lamina Scanner (full 40-µm sections), or using the InCell Analyzer 2200 (GE Healthcare) when using 96-well plates). Analysis was performed using Fiji or Developer software (GE Healthcare, 96-well plates).

Cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 in PBS for 10 min, blocked in BSA 2% for 1 h, washed in PBS, and incubated overnight at 4°C with anti-NFκBiz diluted in 0.1% BSA (rabbit polyclonal, 1:100, cat. 9244 Cell Signaling, Danvers, MA, USA), followed by a 1-h incubation with the secondary antibody Alexa Fluor^® 488 IgG (H+L) conjugate (1:500, cat. A21200, Invitrogen, Carlsbad, CA, USA). Golgi apparatuses were stained with Golgin-97 antibody (monoclonal antibody, 1:200, cat. A21270, Invitrogen) overnight at 4°C and then labeled with a secondary antibody Alexa Fluor^® 594 conjugate (1:500, cat. A21203, Invitrogen) for 1 h at RT. Cell nuclei were stained with DAPI. After washing with PBS, cells were mounted in ProLong^® Gold Antifade Reagent (Life Technologies, Carlsbad, CA, USA) and analyzed with Leica Confocal SP8 in the Advanced Light and Electron Microscopy BioImaging Center (ALEMBIC). Immunohistochemistry (IHC) was performed by the Pathology Unit at IRCCS San Raffaele Hospital using an antibody specific for a long isoform of human SIGIRR on normal kidney tissues and clear cell RCC (ccRCC) tissues.

Differentiated cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature, and then permeabilized by 0.1% triton X-100 (Sigma) for 10 min. After washing and blocking, cells were incubated in the primary antibodies of NKX2.5 (Santa cruz, sc-14033), TNNT2 (DSHB, CT3), α-Actinin (Abcam, ab68167) or β-Catenin (Santa cruz, sc-59737) at 1:100 dilution in 1% BSA at 4 °C overnight. Cells were washed with PBS for three times, and then incubated with alexa 488 conjugated goat anti-mouse antibody (Jackson, 115-545-071) and/or alexa 594 conjugated donkey anti-rabbit antibody (Jackson, 711-585-152) at 1:500 dilution for 1 h at room temperature. After removing the secondary antibodies, the cells were incubated with DAPI for 10 min at room temperature. Finally, the samples were washed three times and then mounted with vectashield (Vector Laboratories). Images were taken using Carl Zeiss Confocal LSM710 or Leica confocal SP8. Sarcomere length was analyzed using Leica Application Suite X (LAS X).