Quantifying immunohistochemical staining of phospho-eIF2α, heme oxygenase-2 and NADPH cytochrome P450 reductase in oligodendrocytes during experimental autoimmune encephalomyelitis

• Published in: Journal of neuroscience methods Vol. 144; no. 2; pp. 227 - 234
• Main Authors: Chakrabarty, Anuradha, Fleming, Kandace K., Marquis, Janet G., LeVine, Steven M.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 15.06.2005
• Subjects: Experimental allergic encephalomyelitis; Heme oxygenase-2; Immunohistochemistry; Multiple sclerosis; NADPH cytochrome P450 reductase; Oligodendrocyte; Phosphorylated eukaryotic initiation factor-2α; Stress response; Immunohistochemistry; Multiple sclerosis; Oligodendrocyte; Heme oxygenase-2; NADPH cytochrome P450 reductase; Phosphorylated eukaryotic initiation factor-2α; Stress response; Experimental allergic encephalomyelitis
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2004.11.010

As a consequence of inflammation associated with multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), stress responses are induced in many cells within the CNS, however, those that occur within the primary pathological target, the oligodendrocyte, are not fully established. Recently, we found that phosphorylated eukaryotic initiation factor-2α (eIF2α), an inhibitor of protein translation associated with the stress response, is expressed in a greater number of oligodendrocytes in EAE animals compared to controls. However, since numerous oligodendrocytes in control animals also expressed phospho-eIF2α, a method was developed to detect expression levels within oligodendrocytes that did not rely on the number of oligodendrocytes that were stained. This method utilized a high dilution of the primary antibody so that the staining density was kept below a maximum plateau which could eliminate expression differences. Furthermore, the staining density within oligodendrocytes, as determined by image analysis, was corrected by the background density or that within neurons. In either case, the density of staining was greater in oligodendrocytes from EAE animals versus controls. The expression of heme oxygenase-2 and NADPH cytochrome P450 reductase also were examined, but unlike phospho-eIF2α, neither was increased in oligodendrocytes from EAE animals compared to controls. In summary, a protocol involving a high dilution of primary antibody and image analysis revealed that the expression of phospho-eIF2α within oligodendrocytes was increased in EAE animals compared to control animals.