title: Improved immunocytochemical identification of neural, endothelial, and inflammatory cell types in paraffin-embedded injured adult rat spinal cord.
abstract: Methods that facilitate the accurate counting of specific neural cell types would be of substantial value in evaluating the efficacy of treatments applied to spinal cord injury. This report describes reliable procedures for identification of neurons, oligodendrocytes, astrocytes, endothelial cells and inflammatory cells (neutrophils and activated macrophage/microglial cells) in paraformaldehyde-fixed, paraffin-embedded injured adult rat spinal cord. Antigen retrieval techniques (enzymatic and thermal) were used to improve antibody access to masked epitopes. To decrease background immunofluorescence and autofluorescence of hemoglobin, the tissue sections were pretreated with 0.1% sodium borohydride in PBS (30min), followed by 1-5min incubation in 0.5% Sudan black in 70% ethanol. Commercially available techniques to amplify the primary signal such as tyramide signal amplification (TSA) and avidin/biotin/peroxidase/DAB/nickel/cobalt amplification (ABP/DABA) were also tested. Hoechst 33342 nuclear staining was used to indicate cell location, number, and integrity, thereby avoiding misidentification of cells. The best antibodies were: anti-NeuN antibody for neurons, anti-S100 for astrocytes, and anti-S100 and APC-7 antibodies in combination for oligodendrocytes, anti-laminin (LN) for endothelial cells, and ED1 antibody for activated macrophages and microglia. Amplification of the primary signal with TSA or ABP/DABA was also found to be beneficial.
authors.affiliation: Institute of Orthopedics and Traumatology of the Hospital of Clinics of the University of São Paulo Medical School, São Paulo, Brazil.
authors.forename: Gizelda T B
authors.initials: GT
authors.lastname: Casella
authors.forename: Mary Bartlett
authors.initials: MB
authors.lastname: Bunge
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Wood
mesh_terms: D000818:Animals; D042783:Endothelial Cells / Q000737:chemistry* / Q000473:pathology; D005260:Female; D007150:Immunohistochemistry / Q000379:methods*; D007249:Inflammation / Q000473:pathology; D009474:Neurons / Q000737:chemistry* / Q000473:pathology; D009504:Neutrophils / Q000737:chemistry* / Q000473:pathology; D016612:Paraffin Embedding / Q000379:methods*; D051381:Rats; D011916:Rats, Inbred F344; D013119:Spinal Cord Injuries / Q000473:pathology*