Pbs nh4cl

For IF analysis 3 × 10⁵ cells were fixed for 10 min in 4% Paraformaldehyde (PFA, Sigma) and washed in 50 mM PBS/NH4Cl (Sigma-Aldrich, Milan, Italy). After washing in PBS 1×, cells were allowed on 35 mm IBIDI μ-Dishes (Ibidi GmbH, Martinsried, Germany) coated with 0.05% poly-L-lysine (Sigma-Aldrich, Milan, Italy) to adhere. Permeabilization was performed with 0.2% Triton/PBS, followed by blocking with 1% BSA/PBS. The seeded cells were immunologically stained with rabbit anti-C15orf41 antibody (1:25) (Atlas Antibodies HPA061023), mouse anti-NUCLEOPHOSMIN (1:200), and secondary antibodies (1:200) (Alexa Fluor 546 anti-rabbit, Life Technologies and Alexa Fluor 488 anti-mouse). Nuclei were stained with 1 μg/ml DRAQ5 in PBS for 15 min at room temperature. Cells were preserved in PBS 1× and imaged using a LEICA TCS SP8 meta confocal microscope, equipped with an oil immersion plan Apochromat 63× objective 1.4 NA. The following settings were used: Green channel excitation of Alexa488 by the argon laser 488 nm line was detected with the 505–550 nm emission bandpass filter. Red channel excitation of Alexa546 by the Helium/Neon laser 543 nm line was detected with the 560–700 nm emission bandpass filter (using the Meta monochromator). Blue channel excitation of DRAQ5 by the blue diode laser 647 nm and emission bandpass filter.

The generated matrices were fixed right after decellularization with formalin 10% (Sigma-Aldrich) for 20 min. Then, samples were treated with a solution of ammonium chloride 50 mM in PBS (NH₄Cl) (Sigma-Aldrich) to reduce the background due to aldehyde groups. Blocking and permeabilization was performed with a solution of 2% BSA and 0.2% Triton X-100 in PBS for 10 min at room temperature. Next, the matrices were stained overnight at 4°C with a combination of rabbit polyclonal anti-Fibronectin antibody (ab2413, Abcam) (1:200) and mouse monoclonal anti-Fibrillin-1 antibody (MAB2499 Millipore) (1:200), either mouse monoclonal anti-Collagen VI antibody (MAB3303, Millipore) (1:400) in PBS with 2% albumin from bovine serum (BSA, Sigma-Aldrich). On the next day, Alexa 568 goat anti-rabbit (A11036, Thermo Fisher Scientific) (1:1000) and Alexa 488 goat anti-mouse (A10667, Thermo Fisher Scientific) (1:1000) were used as a secondary antibody. The incubations were performed for 1 h at room temperature in PBS with 2% BSA. Samples were washed with PBS and mounted with Fluoromount (Sigma-Aldrich) on glass slides. Phalloidin−tetramethyl rhodamine B isothiocyanate (Sigma-Aldrich) was used for F-actin staining (1:1000). Nuclei were stained with Hoechst 33,342 (Molecular Probes) (1:1000). Both dyes were incubated for 1 h at room temperature.