βiii tubulin

The Campenot chamber assay was performed as described28 (link), with minor modifications. In brief, poly-L-ornithine and laminin-coated Aclar embedded coverslips (Electron microscopy sciences) were scratched with a pin-rake (Tyler research), and a three-compartment Teflon divider was placed on silicone grease. Dissociated sensory neurons from E13 dorsal root ganglia (DRG) were plated in the central compartment using medium supplemented with 10 ng/ml human NGF. The distal compartments were filled with medium containing 75 ng/ml human NGF. The next day, cultures were treated with 5 μM cytosine arabinoside. On days 5 and 7, cultures were infected with the corresponding lentiviruses expressing scrambled, XIAP or FAIM-L shRNAs and EMPTY EGFP or FAIM-L EGFP overexpression vectors, respectively. To trigger local axonal degeneration, NGF-containing medium from axonal compartments was replaced with medium containing sheep anti-NGF 1:50 (Abcam). Cultures were fixed with 4% paraformaldehyde 24 h after NGF removal and processed for βIII-Tubulin (Covance) (1:4000) and GFP (Life technologies) (1:500) immunofluorescence. Alternatively, cells were collected for Western blot analysis.

Differentiated cultured NS were re-suspended in ice-cold cell lysis buffer (Cell Signaling Technology) with protease inhibitor cocktail (Roche) and incubated for 15–30 min on ice. A total amount of 30 µg of protein was loaded on a 10% or 12% SDS-PAGE gel and transferred to nitrocellulose membranes (Protran, Whatman). The membranes were blocked in Tris-buffered saline with 0.05% Tween-20 and 5% skimmed milk or 4% BSA (MAP-2 blots), incubated with primary and secondary antibodies, and washed according to standard procedures. Primary antibodies used were: musashi-1 (1/500, rabbit; Abcam), nestin (1/500, rabbit; Abcam), SOX-2 (1/1000, mouse, Cell Signaling), ki67 (1/500, rabbit, Abcam), PCNA (1/500, mouse, Millipore), β-III-tubulin (1/1000, TuJ clone; mouse; Covance), MAP-2 (1/500, mouse; Sigma), CNPase (1/500, rabbit, Covance), GFAP (1/1000, mouse; Sigma) and α-tubulin (1/5000, mouse; Sigma). Secondary peroxidase-conjugated (1/1000) donkey anti-rabbit (Amersham Biosciences, GE Healthcare), or rabbit anti-mouse antibodies (Jackson Immunoresearch) were used. Values in figures are the average of the quantification of at least three independent experiments each of them corresponding to four different cellular pools.

Plated cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. Cells were washed three times with PBS after each step and then blocked with 5% bovine serum albumin (Nacalai Tesque) for at least 1 h at room temperature. The cells were permeabilized prior to primary antibody incubation. Cells were then incubated with primary antibodies at 4 °C overnight and washed three times with PBS. The following primary antibodies were used in this assay: NANOG (1:500; ReproCELL, Yokohama, Japan), SSEA4 (1:1,000; Chemicon, Darmstadt, Germany), βIII-tubulin (1:2,000; Covance, Princeton, NJ), MAP2 (1:1,000; Millipore, Billerica, MA), SMI-32 (1:2,000; Covance), GFAP (1:1,000; DAKO, Glostrup, Denmark), TFG (1:1,000; Protein Tech, Rosemont, IL), Ubiquitin (1:1,000; DAKO), FK2 (1:1,000; MBL, Nagoya, Japan), SOX17 (1:1,000; R&D Systems, Minneapolis, MN), and αSMA (1:500; DAKO). Suitable secondary antibodies (Invitrogen) were incubated with samples at room temperature for 1 h, and washed three times with PBS. Nuclear staining was also performed with 0.5 g/mL 4′,6-Diamidino-2-Phenylindole (DAPI) for 5 min, and then the cells were washed three times with PBS. Coverslips were mounted in ProLong Gold antifade reagent (Invitrogen). Images were collected using LSM710 (Carl Zeiss, Oberkochen, Germany) or IN Cell Analyzer 6000 (GE Healthcare).

Cells were fixed in 4% paraformaldehyde for 30 min at room temperature, washed with PBS, and permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room temperature, followed by blocking for 30 min with Block Ace (Yukijirushi, Tokyo, Japan). After incubation with primary antibodies overnight at 4 °C, cells were washed three times with PBS and incubated with appropriate secondary antibodies for 1 h at room temperature. Cell images were acquired with Delta Vision (GE Healthcare, Chicago, IL) or IN Cell Analyzer 6000 (GE Healthcare). The numbers of cells were quantified with IN Cell Analyzer 6000 and IN CELL Developer toolbox software 1.9 (GE Healthcare). The following primary antibodies were used in this assay: βIII tubulin (1:2,000, Covance, Princeton, NJ), NeuN (1:500, Millipore, Darmstadt, Germany), TOC1 (1:1,000), Nanog (1:200, Abcam, Cambridge, UK), and SSEA4 (1:500, Millipore).

Differentiated cells were fixed with 4% paraformaldehyde (PFA) for 20 min at 4°C. Fixed cells were washed three times with Tris buffered saline (TBS). Non-specific binding was blocked and the cells were permeabilized with 0.02% Triton X-100 and 5% normal goat serum (NGS) (PAA, The Cell Culture Company, Somerset, UK), for 1 h at room temperature (RT). Primary antibodies diluted in 1% NGS blocking buffer were added to the cultured cells overnight at 4°C (Oct4, 1:100, Santa Cruz; β-III-tubulin 1:500, Covance; TH, 1:1000, Chemicon; Serotonin, 1:500, Abcam). Negative primary controls consisted of cells treated with blocking buffer without the addition of primary antibodies. On the next day, three TBS washes were applied to the cells followed by incubation with Alexa Fluor secondary antibodies, (Cheshire Sciences, UK) diluted to 1:300 in 1% NGS blocking buffer, for 2 h at RT. Cultures were washed three times with TBS. Coverslips were mounted onto microscope slides using Vectashield hardset mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) (Vector Labs, Peterborough, UK) to counterstain cell nuclei, before visualisation the following day.

For immunofluorescence staining, cells were fixed in 4% paraformaldehyde for 10 min at room temperature. After PBS washing, cells were permeabilized with 0.3% Triton X-100 for 45 min at room temperature. After removal of the Triton X-100 solution, cells were washed with PBS and stained at 4°C overnight with primary antibodies at appropriate dilutions. Primary antibodies used include Tra-1-81 (1∶200, eBioscience, San Diego, CA), Tra-1-60 (1∶100, Stemgent, Cambridge, MA), Oct4 (1∶100, Stemgent, Cambridge, MA), Sox2 (1∶100, Neuromics, Minneapolis, MN), β III-tubulin (1∶2000, Covance, Princeton, NJ), HB9 (1∶10, Hybridoma Bank, Iowa City, Iowa). Cells were stained at room temperature with the secondary antibody for 2 hours at 1∶800 dilution, including Cy3-conjugated goat anti-rabbit IgG (Chemicon, Temecula, CA) or Cy3-conjugated rabbit anti-mouse IgG antibody (Millipore). Nuclei were stained with DAPI (Life Technology, Carlsbad, CA) at a concentration of 1 μg/ml.