To examine apoptosis signaling in neurons with axons undergoing very delayed resealing, DTMR was applied 24 h after the initial TX, and the lampreys were allowed to recover for 2 (n = 8), 4 (n = 4), and 10 (n = 3) weeks after injury. Afterwards, freshly dissected brains were processed using the Image-iT™ LIVE Green Poly Caspases Detection Kit (I35104, Invitrogen, Carlsbad, CA, USA), which uses the FLICA reagent, FAD-VAM-FMK, to label activated caspases. As previously reported, apoptotic lamprey RS neurons were labeled as early as 1 week after TX (peaking at 4 weeks), while these neurons did not become positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) until 4 weeks after injury (peaking at 12–16 weeks) [31 (link)]. After processing, the brains were fixed in PFA, washed thoroughly with PBS, and imaged by fluorescence microscopy.
In the analysis of these assays, RS neurons in each recovery group were binned according to the combinations of dye they contained: i.e., DTMR only (not sealed, not apoptotic), FLICA only (sealed, apoptotic), and DTMR + FLICA (not sealed, apoptotic). The percent labeling after TX was calculated as: (number of labeled neurons in a bin divided by the total number of cells in the group) × 100.
In the analysis of these assays, RS neurons in each recovery group were binned according to the combinations of dye they contained: i.e., DTMR only (not sealed, not apoptotic), FLICA only (sealed, apoptotic), and DTMR + FLICA (not sealed, apoptotic). The percent labeling after TX was calculated as: (number of labeled neurons in a bin divided by the total number of cells in the group) × 100.
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