Clone 6 11b 1

Cultured colonies were fixed with 10% neutral-buffered formalin in overnight at room temperature. After rinsing with 70% ethanol, fixed colonies were immobilized with Histogel (Thermo Scientific) for paraffin embedding. Sectioned embedded colonies were stained with haematoxylin and eosin and immunostained with antibodies for CCSP (1:200, Santa Cruz clone T-18), pro-SPC (1:200, Santa Cruz clone FL-197), p63 (1:200, Biocare clone 4A4), acetylated-tubulin (1:1,000; Sigma, clone 6-11B-1). Secondary antibody staining included donkey a-mouse 488, donkey a-rabbit 594, donkey a-goat 647 (1:400, Invitrogen) and Prolong Gold with DAPI (Life Technologies).

Fixed cells or frozen tissue sections were blocked and permeabilized with PBS containing 4% bovine serum albumin, 2% goat serum, 100 mM glycine and 0.3% Triton X-100. Primary antibody was applied overnight at 4 °C diluted in blocking solution, washed 3 times in PBS, incubated for 2 h in the secondary antibody at room temperature, with DAPI, then washed four times in PBS, mounted in Vectashield® and imaged with confocal microscope Zeiss LSM800 or imaged with conventional microscope Zeiss Axiovert 135 M equipped with the camera Leica DC 350 FX CCD monochrome. The following antibodies were used: anti-acetylated tubulin (1/400, clone 6-11B-1, Sigma), anti p62 (1/200, GP62-C, Progen, Germany), anti-Ubiquitin conjugated protein1 (1/100, BML-PW8810, Enzo), anti - Lc3b (1/100, #2775, Cell Signaling Technology, Danvers), anti-beta3 tubulin (1/500, TUJ1, Biolengend), anti-NEFM (1/500, Poly28410, Biolegend), anti-NEFH (1/2000, Smi32, Biolegend), anti-cleaved caspase 3 (1/100; 5A1E, Cell Signaling Technology, Danvers).

The tails were fixed in 4% paraformaldehyde (PFA) or MEMFA (0.1 M MOPS pH7.4, 2 mM EGTA, 1 mM MgSO₄, 3.7% formaldehyde) and dehydrated in methanol. Rehydrated samples were embedded in 25% fish gelatin / 20% sucrose and cryosectioned at 12 μm thickness. For non‐regenerating spinal cords, sections were taken at least 250 µm anterior to the amputation plane. For the regenerate, only sections posterior to the amputation plane and anterior to the neural ampulla were considered. The following antibodies were used: rabbit anti‐Sox3 (1:500 at 3 dpa or 1:1,000 at 0 and 5 dpa) and anti‐Myt1 (1:750) were a gift from Nancy Papalopulu; mouse anti‐PCNA was used at 1:500 (PC10, Sigma); rabbit anti‐cleaved caspase 3, Asp175 (1:100, 9661, Cell Signalling Technology), rabbit anti‐pH3 (1:100, 06‐570, Sigma), mouse anti‐BrdU (1:300, clone MoBU‐1, B35128, Invitrogen) and anti‐Acetylated Tubulin (1:1,000, clone 6‐11B1, Sigma). Secondary antibodies were goat anti‐rabbit IgG, Alexa Fluor 488 (A‐11008, Invitrogen) and goat anti‐mouse IgG, Alexa Fluor 568 (A‐11004, Invitrogen).
Whole‐mount in situ hybridisation on embryos and tails was performed as previously described (Harland, 1991 (link)). The foxm1 probe was generated from the TGas064p23 clone linearised with ClaI and transcribed using T7 polymerase.