Activated caspase detection in living tissue combined with subsequent retrograde labeling, immunohistochemistry or in situ hybridization in whole-mounted lamprey brains.

In the lamprey brain, there are 18 pairs of identified spinal-projecting neurons whose regenerative abilities have been characterized. The "bad-regenerating" neurons show a very delayed form of apoptosis after axotomy (Shifman et al., 2008). Theoretically, this should provide a long window of opportunity to intervene therapeutically, so it would be helpful if we could identify the early stages of this process in vivo. Until now, there has been no method to link mRNA or protein expression directly to early-stages neuronal apoptosis in vivo. Here we describe a double-labeling protocol in whole-mounted lamprey brain for simultaneous detection of early stage apoptosis, using Fluorochrome-Labeled Inhibitors of Caspases (FLICA), and either mRNA, using in situ hybridization, or protein expression, using immunohistochemistry. To improve brain preservation, the working temperature during the FLICA stage was lowered from 37°C to 4°C (Barreiro-Iglesias and Shifman, 2012). Using this method, neurofilament protein was demonstrated by immunohistochemistry in neurons previously reacted by FLICA. The method also revealed that mRNA for the receptor protein tyrosine phosphatase PTPσ is expressed selectively in FLICA-positive neurons. In addition, our study showed that a retrograde labeling technique can be used in the context of FLICA labeling. FLICA label colocalized with TUNEL staining, confirming that FLICA labeling is a reliable marker of apoptosis in lamprey brain. Our results suggested that we can combine caspase detection with other techniques in vivo to investigate the roles and mechanisms of activated caspases and other molecules in retrograde cell deaths and regenerative abilities of neurons.