2.3. Immunofluorescence and Immunohistochemical Assays

Cells were fixed with 3.7% formaldehyde for 30 min at room temperature (RT) followed by permeabilization with 0.1% Triton X-100 for 5 min and blocking with 1% Bovine Serum Albumine (BSA) for 1h. For type 1 and type 3 Collagen staining, cells were incubated overnight at 4 °C with a mouse monoclonal anti-type 1 Collagen antibody (1:100, Sigma-Aldrich) and a rabbit polyclonal anti-type 3 Collagen antibody (1:100, Santa Cruz Biotechnology). Following incubation with the primary antibody, cells were incubated for 1h at RT with the DyLight 649 anti-mouse IgG (1:500, BioLegend, CA) and the Alexa FluorTM 488 goat anti-rabbit IgG (1:500; Thermo Fisher Scientific, USA). Cell nuclei were stained with DAPI solution (1:1000) for 5 min. Images acquisition was at 20× magnification on a fluorescent microscope (Eclipse Ti-E Inverted Microscope; NIKON Instruments Inc., USA).

For 3D scaffold immunohistochemical analysis, slices were permeabilized with 0.1% Triton X-100 for 5 min, and non-specific staining blocked with 1% BSA for 1h at RT. For type 1 Collagen staining, slices were incubated overnight at 4 °C with a rabbit polyclonal anti-type 1 Collagen antibody (1:200, AbCam). Following incubation with the primary antibody, slices were incubated for 1h at RT with Alexa Fluor^TM 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, USA) antibody. Subsequently, cell nuclei were stained with DAPI solution (1:1000) and incubated for 5 min. Images were acquired as described above. Image quantification was performed using image analysis software (ImageJ, National Institutes of Health, USA) [56,57] by measuring the red and green areas where type 1 and type 3 collagen, respectively, are expressed. A minimum of 10 image fields was used for the image analysis at each time point. Signal intensity at each time point was normalized by the cell number (e.g., by amount of cell nuclei revealed by DAPI staining).

Sirius red staining was performed using the Picrosirius Red Stain Kit (Polysciences, Inc., USA). Sections of 15 μm of thickness were stained in hematoxylin for 8 min, then washed in water for 2 min. The sections were dipped into phosphomolybdic acid for 2 min, then washed in water for 2 m. Then they were dipped into Picrosirius Red F3BA Stain for 60 min and dipped into HCl 0.1M solution for 2 min. The sections were dehydrated in increasing ethanol gradient solutions (70–75–95–100%) and finally dipped into xylene for 5 min. Eukitt medium was used to mount the samples.