Mouse anti βiii tubulin antibody

The cells were fixed in ice-cold methanol for 5 minutes and then washed 4 times in PBS followed by a blocking incubation with skimmed milk (2 g/ml) in PBS for 30 minutes. After blocking, the coverslips were incubated with primary antibody diluted in the blocking solution overnight at 4°C: mouse anti-βIII tubulin antibody (1:2000, Promega) combined with one of the anti-integrin antibodies (α1, α3, α5, αV, β1, β3) at the dilution indicated in Table 1.
The following day, the coverslips were washed and incubated with a secondary biotinylated anti-rabbit antibody, (1:1000, Vector) for 30 minutes, washed three times and followed by 45 minutes incubation with Streptavidin-Alexa Fluor 568 (1:500, Vector) to stain the different integrins and Alexa Fluor donkey anti-mouse 488 (1:500, Invitrogen) to stain the βIII-tubulin positive RGCs. Coverslips were then washed in PBS, followed by nuclear counterstaining with 4,6-diamidino-2-phenylindole (1:10,000, DAPI, Sigma). After a final wash in PBS, coverslips were mounted in Fluorsave (Calbiochem/Merck Chemicals, Beeston, UK).

NG108-15 neuronal cells, and primary Schwann cells, were immunolabelled for specific proteins to visualize neurite outgrowth from neuronal cells and confirm Schwann cell phenotype, respectively, as previously described [24 (link), 27 ]. Briefly, samples were fixed with 3.7% (v/v) paraformaldehyde (20 min), permeabilized with 0.1% Triton X-100 (20 min) and blocked with 3% bovine serum albumin (BSA), in PBS (30 min) [24 (link), 27 ].
NG108-15 neuronal cells were labelled with a mouse anti-β III-tubulin antibody (1:250) (Promega, UK), for 48 h at 4°C, followed by a Texas Red-conjugated anti-mouse IgG antibody (1:200 dilution in 1% BSA from Vector Labs, USA), for 90 min at room temperature, to visualize and measure neurite outgrowth [24 (link), 27 ].
Schwann cells were identified with a polyclonal rabbit anti-S100β (1:250) (Dako, Denmark) antibody, incubated for 48 h at 4°C, followed by a FITC-conjugated secondary anti-rabbit IgG antibody (1:100 dilution in 1% BSA), for 90 min at room temperature [24 (link), 27 ]. All cells were incubated with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Aldrich) (300 nM) for 30 min, at room temperature to observe cell nuclei [27 ]. Cells, and samples, were imaged using an upright Zeiss LSM 510 confocal microscope using three fields of view for quantitative analysis [24 (link), 27 ].

SH-SY5Y cells were seeded in 12-well plates (1 × 10⁵ cells/well), previously coated with the different type of collagen films, and cultured for 9 days in complete medium. Then, cells on collagen films were fixed for 20 min with 10% formalin solution, incubated for 25 min with gelatin dilution buffer 1× (GDB; 0.4% (w/v) gelatin, 40 mM sodium phosphate buffer, pH 7.2, 0.9 M NaCl, 0.2% (v/v) Triton X-100) to perform permeabilization and blocking and incubated with mouse anti β-III tubulin antibody (1:250; Promega Italia Srl, Milan, Italy) in GBD 1× for 2 h at room temperature. After washes, cells were incubated with Texas Red-conjugated goat anti-mouse IgG (1:200; ThermoFisher Scientific) in GDB 1× for 1 h at room temperature. Four μM Hoechst 33342 (ThermoFisher Scientific) was used to stain nuclei. Finally, the collagen films were mounted on glass slides using ProLong Gold Antifade Reagent (ThermoFisher Scientific). Images were acquired using an inverted confocal laser scanning microscope equipped with a Plan-Neofluar 63 × 1.4 oil objective (Carl Zeis Meditec AG, Jena, Germany). Excitation was performed using an Ar-laser diode (540 nm) and an ultraviolet 25 mV laser diode (405 nm). The pinhole was set to 1 AU.