Image it live green poly caspases detection kit

The Image-iT LIVE Green Poly Caspases Detection Kit (Cat. number I35104, Invitrogen, USA), Image-iT LIVE Green Caspase-3 and -7 Detection Kit (Cat. number I35106, Invitrogen), and the Image-iT LIVE Green Caspase-8 Detection Kit (Cat. number I35105, Invitrogen) were used to detect activated caspases in identified reticulospinal neurons of larval sea lampreys after a complete SC transection. These kits contain 1 vial (component A of the kit) of the lyophilized FLICA reagent (FAM-VAD-FMK for the detection of all activated caspases, FAM-DEVD-FMK for the detection of activated caspase-3 and caspase-7, and FAM-LETD-FMK for the specific detection of activated caspase-8). Experiments were done as previously described [30 (link), 31 (link)]. Experiments for the detection of all activated caspases were performed in animals 2 weeks after the complete SC transection (n = 5). Experiments for the detection of activated caspase-8 were in animals 4 weeks (n = 4) after the complete SC transection. Experiments for detection of activated caspase-3 and caspase-7 were done in control unlesioned animals (n = 5) and in animals 6 weeks (n = 3) and 10 weeks (n = 4) after the complete SC transection.

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.