title: Activated caspase detection in living tissue combined with subsequent retrograde labeling, immunohistochemistry or in situ hybridization in whole-mounted lamprey brains.
abstract: 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.
authors.affiliation: Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation), 3500 North Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Jianli
authors.initials: J
authors.lastname: Hu
authors.forename: Guixin
authors.initials: G
authors.lastname: Zhang
authors.forename: Michael E
authors.initials: ME
authors.lastname: Selzer
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001921:Brain / Q000187:drug effects / Q000201:enzymology*; D061945:Caspase Inhibitors / Q000494:pharmacology; D020169:Caspases / Q000032:analysis* / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D020287:In Situ Nick-End Labeling; D007798:Lampreys
chemical_list: D061945:Caspase Inhibitors; D020169:Caspases
keywords: Apoptosis; Brain; Caspase; Lamprey; Wholemount immunohistochemistry; Wholemount in situ hybridization
references.citation: Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11686-90
references.pmid: 8524829
references.citation: Exp Neurol. 1994 May;127(1):94-105
references.pmid: 7515355
references.citation: Cell Cycle. 2002 Mar-Apr;1(2):124-31
references.pmid: 12429921
references.citation: J Comp Neurol. 2008 Sep 20;510(3):269-82
references.pmid: 18634003
references.citation: Nat Rev Neurosci. 2004 Sep;5(9):686-700
references.pmid: 15322527
references.citation: Nat Neurosci. 2011 May;14(5):545-7
references.pmid: 21441921
references.citation: Nat Rev Neurosci. 2012 May 18;13(6):395-406
references.pmid: 22595785
references.citation: J Neuropathol Exp Neurol. 2009 Jul;68(7):757-61
references.pmid: 19535996
references.citation: Nat Rev Mol Cell Biol. 2007 May;8(5):405-13
references.pmid: 17377525
references.citation: Nat Rev Mol Cell Biol. 2004 Nov;5(11):897-907
references.pmid: 15520809
references.citation: Nat Rev Mol Cell Biol. 2008 Jul;9(7):532-42
references.pmid: 18568041
references.citation: J Neurosci. 1997 Jul 1;17(13):5206-20
references.pmid: 9185558
references.citation: J Neurochem. 2006 May;97(4):1104-10
references.pmid: 16573645
references.citation: Nat Protoc. 2006;1(4):1725-31
references.pmid: 17487155
references.citation: Neurosci Lett. 1984 Jul 27;48(2):145-8
references.pmid: 6483278
references.citation: Exp Neurol. 1994 Apr;126(2):256-69
references.pmid: 7523177
references.citation: Cell. 2010 May 28;141(5):859-71
references.pmid: 20510932
references.citation: Enzyme Res. 2012;2012:835731
references.pmid: 22829997
references.citation: J Comp Neurol. 1993 Oct 8;336(2):194-210
references.pmid: 8245215
references.citation: Nat Neurosci. 2011 Jan;14(1):69-76
references.pmid: 21151119
references.citation: Annu Rev Biochem. 2004;73:87-106
references.pmid: 15189137
references.citation: Neuron. 2006 Aug 3;51(3):283-90
references.pmid: 16880123
references.citation: Cytometry A. 2007 Aug;71(8):536-7
references.pmid: 17640078
references.citation: J Clin Invest. 2005 Oct;115(10):2665-72
references.pmid: 16200200
references.citation: Nat Neurosci. 2006 Oct;9(10):1234-6
references.pmid: 16980964
references.citation: Cell Death Differ. 2006 Sep;13(9):1423-33
references.pmid: 16676004
references.citation: Cytometry A. 2007 Aug;71(8):605-11
references.pmid: 17549763