Ab8874

The polycolonal antibodies for Lingo-1 (ab23631), NgR (ab26291), p75 (ab8874), TROY (ab12126), and Myt1 (ab82844), and monoclonal antibody for WNK1 (ab128858) were all purchased from Abcam (Melbourne VIC, Australia). Primary antibody dilution ranged from 1:200 to 1:500. Secondary antibodies for rabbit (AP307P) and mouse (AP308P) were purchased from Merck Millipore (Bayswater, VIC, Australia) and were used at a concentration of 1:3000. The monoclonal antibody for β-actin (MAB1501) was also purchased from Merck Millipore and was used at a concentration of 1:5000. Antibody specificity has been previously demonstrated by the use of appropriate positive controls as documented in both the literature [33 (link),39–44 (link)] and the antibody datasheets provided by Abcam.

The proper p75+/GFAP+ phenotype of olfactory ensheathing cells was verified at day five of the culture on the biomaterials. For immunofluorescence staining, cells were fixed in 4% paraformaldehyde for 15 min at room temp. After fixation, cells were washed three times in PBS, and permeabilized using 0.05% triton x-100 solution in PBS for 20 min. After triple washing in PBS, cells were incubated with primary antibodies (anti-GFAP produced in rabbit, ab7260, 1:1000; anti-p75 NGF receptor produced in rabbit, ab8874, 1:1000; all from Abcam, Cambridge, UK) for one hour at 37 °C. After triple washing in PBS containing 2% of FBS, cells were incubated with secondary antibodies (goat anti-rabbit IgG conjugated to atto-488 fluorophore, Sigma, 1:400) for one hour at 37 °C. After incubation, cells were washed three times in PBS and counterstained with 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, 5 µg/mL) for 5 min. Preparations were observed by means of inverted fluorescence microscope (AxioObserver A1, Zeiss), and documented with a digital camera (PowerShotA630, Canon, Tokyo, Japan). Pictures were merged using ImageJ software.

IF and IHC was performed overnight keeping the slides in humified chambers^{11 (link)} and incubating them with the following primary antibodies: ΔNp63 (ab172731, Abcam, 1:500), GFP (ab13970, Abcam, 1:200), Ki67 (ab15580, Abcam, 1:1,000), cytokeratin 5 (ab53121, Abcam, 1:250), acetylated tubulin (T7451, Sigma-Aldrich, 1:200), mucin 5ac (MA1-21907, Thermo Fisher Scientific, 1:100), cleaved caspase 3 (9661, Cell Signaling, 1:200), CC10 (sc-25555, Santa Cruz, 1:200), SPC (sc-7705, Santa Cruz, 1:100) and NGFR (ab8874, Abcam, 1:100).