Blocking buffer

Costar high-binding immunoplates (96 wells) were incubated overnight with 50 µL of the recombinant C0C2 domain of human cMyC, the relevant peptides or a 28mer peptide of the C5 domain specific for human cMyC in (2 µg/mL TBS, pH 7.4) per well at RT. After blocking non-specific binding sites with blocking buffer (Roche) for 2 h, wells were washed three times with NaCl (0.154 M) containing Tween 20 (0.05 %). Hybridoma supernatants (50 µL) were then added to each well for 2 h. After extensive washing with NaCl-Tween, bound antibodies were detected by incubating the plates with a goat anti-mouse IgG, Fc-specific and conjugated to HRP, (Dianova, Hamburg, Germany) at a dilution of 1:10,000. Plates were read 10–15 min after adding the HRP substrate ABTS (Roche).

Cryosections of mouse or zebrafish brains on slides were pre-warmed at RT in TBS solution and then washed three times in fresh TBS to remove all O.C.T., leaving behind the sections on the slides. Staining was performed as follows: sections were incubated 30 min in 1X Tris Glycine, then 1 hr in 0.1% Triton X-100 and finally 1 hr in blocker (1% Roche Blocking Buffer, 5% Sheep Serum in TBS-T). Samples were incubated overnight at 4°C in blocker with primary antibody (GFP 1:2000 and CD206 at 1:1000). Samples then were washed in TBS-T, four or more times as needed and incubated in blocker with secondary antibody for up to 2 hr at room temperature and washed in TBS-T at least four times.

Nuclei exhibiting apoptotic changes were identified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using the in situ cell death detection kit, TMRred (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s recommendations. Briefly, the sections were rehydrated in PBS with 0.1% Tween 20 and then fixed in 4% paraformaldehyde at room temperature for 20 min. After several washes with T-PBS, the sections were fixed in 0.1% (w/v) sodium citrate (0.1% C₅H₃O₇Na₃·2H₂O and 0.1% Triton X-100) at room temperature for 2 min. After washing, the sections were immunoreacted with anti-laminin antibody (1:500, Dako Japan, Tokyo, Japan) diluted in 1% (w/v) blocking reagent (Roche) in maleic acid buffer (0.1 mol/L maleic acid and 0.15 mol/L NaCl, pH 7.5) at room temperature for 1 h. After several washes in T-PBS, a fluorescein isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody (1:250, Sigma) in Blocking buffer (Roche) was applied for 1 h at room temperature. After several washes in T-PBS, the TUNEL reaction mix diluted in TUNEL Dilution Buffer (Roche) was applied and incubated at 37°C for 60 min in the dark. After several washes in T-PBS, the sections were cover slipped using Vectashield mounting medium with 4′, 6-diamidino-2- phenylindole (Vector Labs, Burlingame, CA).