# Poly(vinyl alcohol)/Gelatin Scaffolds Allow Regeneration of Nasal Tissues
## 2 Materials and Methods
### 3D Model Characterization  
#### 2.9.1 Histological Characterization of 3D Models  
After formalin fixation, constructs employed to the creation of chondrogenic, vascularized osteogenic and fibrocartilaginous models were dehydrated with a graded series of ethanol (from 70% to 100%). After 3 h of incubation in absolute ethanol (Bio-Optica), samples were clarified in xylene (two steps of 45 min each) (Sigma-Aldrich). All steps were performed in a thermostatic bath at 40 \({}^{\circ}\)C. Successively, specimens were rinsed in liquid paraffin (Histoplast LP, Thermo Fisher Scientific, Waltham, MA, USA) at 60 \({}^{\circ}\)C for 2 h and paraffin-embedded. Sections 5 \(\upmu\)m thick were obtained by standard micromote and mounted onto glass slides. Before each histological staining or immunoreaction, sections were deparaffined in xylene (two steps of 7 min each), rehydrated in absolute ethanol (three steps of 7 min each) and rinsed in distilled water for 5 min. Before each staining, sections of both seeded PVA and chondrogenic pellet were deparaffinized; after each staining, the sections were dehydrated. * Hematoxylin and Eosin (H&E) staining  
Sections were deparaffinized rinsing them in xylene twice for 7 min, then they were rehydrated by absolute ethanol three times for 7 min and washing them in distilled water for 5 min. Samples were then stained with Mayer's hematoxylin for 5 min, washed in tap water for 5 min and counterstained with 1% _w/v_ eosin for 1 min. After washing in distilled water, specimens were dehydrated by three washes in absolute ethanol and clarified by three washes in xylene (5 min for each step), then they were mounted with a coverslip by DPX mounting agent. * Alcian Blue pH 1 staining  
Specimens were incubated in Alcian Blue pH 1 solutions for 30 min and in revealing solutions for 10 min, according to manufacturer's instructions. After the incubation time, sections were washed in distilled water, counterstained with a distilled water solution containing 0.1% _w/v_ Nuclear Fast Red and 5% _w/v_ aluminum sulfate for 5 min and washed in tap water for 5 min. Sections were dehydrated and mounted as previously described. * Von Kossa staining  
Deparaffinized sections were incubated with 1% _w/v_ silver nitrate exposed to light for 15 min, 0.5% _w/v_ Pyrogallol for 2 min and 5% _w/v_ sodium thiosulphate for 2 min. All the solutions were made in distilled water. After each passage, washings in distilled water were performed. The counterstainedaining was performed by incubating cells with 0.1% _w/v_ nuclear fast red diluted in a distilled water solution containing 5% _w/v_ aluminum sulphate, for 5 min and washing in tap water for 5 min in order to reveal the reaction. Sections were dehydrated and mounted as previously described. * Immunohistochemical analysis Sections employed for the detection of aggrecan and collagen type II were incubated in citrate buffer in a thermostatic bath at 90 \({}^{\circ}\)C for 30 min, in order to unmask antigens, then they were cooled down for 30 min and washed in 1\(\times\) PBS for 10 min; cells and sections employed for the detection of the other antigens were permeabilized with 0.2% _w/v_ Triton X-100 in 1\(\times\) PBS for 10 min. All specimens were incubated in a methanolic solution containing 0.06% _v/v_ H\({}_{2}\)O\({}_{2}\) in the dark for 15 min to quench peroxididases. Sections were rinsed in distilled water and washed in 1\(\times\) PBS. Therefore, specimens were incubated with 5% _v/v_ goat serum diluted in 1x PBS for 20 min at 37 \({}^{\circ}\)C to block specific binding sites of the secondary antibodies. Specimens were washed in 1x PBS and then incubated with primary antibodies diluted in 0.1% _w/v_ BSA/1x PBS solution in a moist chamber overnight at 4 \({}^{\circ}\)C. The following antibodies with specified concentrations were used: anti-aggrecan 1:50; anti-Sox-9 1:100; anti-collagen type II 1:50, anti-collagen type I 1:1000, anti-ALP 1:100, anti-Osteoclacin 1:400, anti-TGF\(\theta\)-1 1:100, anti-Flk-1 1:50, anti-p-Flk-1 1:50, anti-PECAM-1 1:100.