Exposure of nuclear antigens in formalin-fixed, paraffin-embedded necropsy human spinal cord tissue: detection of NeuN.
Immunohistochemical and immunofluorescence staining approaches are powerful tools for characterization of the endogenous protein expression and subcellular compartmentalization. However, several technical problems hamper identification of low-abundance nuclear proteins in archival formalin-fixed, paraffin-embedded human neural tissue. These include loss of protein antigenicity during tissue fixation and processing, and intrinsic auto-fluorescence associated with the tissue related to its fixation and the presence of lipofuscin. We evaluated several antigen retrieval methods to establish a strategy for detection of neuronal nuclear proteins in human spinal cord formalin-fixed, paraffin-embedded tissue. Thus, using immunostaining of the neuron-specific nuclear protein NeuN as the outcome measure, we found that heating tissue sections in an alkaline pH buffer unmasked protein epitopes most effectively. Moreover, staining by immunohistochemistry with diaminobenzidine tetrahydrochloride chromagen was superior to immunofluorescence labeling, likely due to the signal amplification steps included in the former approach. Auto-fluorescence in the tissue sections can be effectively reduced, but a sufficient fluorescence signal associated with specific antibody labeling could not be detected above this background for NeuN in the nucleus.
Gill
Sandeep K
SK
Department of Physiology and Pharmacology, Medical Sciences Building, University of Western Ontario, London, Ont., Canada N6A 5C1. gsandeep@fmd.uwo.ca
Ishak
Margaret
M
Rylett
R Jane
RJ
D000818:Animals; D034961:Antigens, Nuclear / Q000032:analysis*; D001391:Azo Compounds / Q000494:pharmacology; D001881:Borates / Q000494:pharmacology; D004396:Coloring Agents / Q000494:pharmacology; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles / Q000378:metabolism; D051379:Mice; D009281:Naphthalenes; D009336:Necrosis; D009419:Nerve Tissue Proteins / Q000032:analysis*; D016612:Paraffin Embedding / Q000379:methods*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D051381:Rats; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D016707:Tissue Fixation / Q000379:methods*
D034961:Antigens, Nuclear; D001391:Azo Compounds; D001881:Borates; D004396:Coloring Agents; D005404:Fixatives; D007211:Indoles; D009281:Naphthalenes; D009419:Nerve Tissue Proteins; C506808:neuronal nuclear antigen NeuN, human; C080440:sodium tetraethylborate; D005557:Formaldehyde; C007293:DAPI; C016118:Sudan Black B; D010751:Phosphopyruvate Hydratase