TY - JOUR
T1 - A specific and sensitive method for visualization of tumor necrosis factor in the murine central nervous system
AU - Lambertsen, K L
AU - Drøjdahl, N
AU - Owens, T
AU - Finsen, B
PY - 2001/6/1
Y1 - 2001/6/1
N2 - We present here sensitive, simple and robust methods for detection of tumor necrosis factor (TNF) mRNA and TNF in histological sections and homogenates of brain tissue from mice subjected to focal cerebral ischemia or hippocampal axonal lesioning. Both types of lesions are characterized by induction of TNF synthesis in resident microglial cells, which in the ischemic lesions are supplemented by TNF synthesizing, blood-borne macrophages. In situ hybridization for TNF mRNA is performed using alkaline phosphatase-labelled oligodeoxynucleotide probes. These probes show excellent rendition of individual cells, and can successfully be combined with immunohistochemical procedures. We also describe a sensitive immunohistochemical method for detection of TNF, which can be combined with visualization of an additional antigen. The specificity of the histological procedures are confirmed by RT-PCR and Western blot analysis on homogenates prepared from microdissected brain regions. Advantages and disadvantages of the methods are discussed with emphasis on the specificity and sensitivity of the histological procedures. Our strategy for detection of TNF mRNA and protein provides a solid basis for clarifying the cellular synthesis, regulation and function of TNF in the normal, injured or diseased CNS. Furthermore, the methodology can readily be applied in studies of other cytokines and growth factors in the CNS.
AB - We present here sensitive, simple and robust methods for detection of tumor necrosis factor (TNF) mRNA and TNF in histological sections and homogenates of brain tissue from mice subjected to focal cerebral ischemia or hippocampal axonal lesioning. Both types of lesions are characterized by induction of TNF synthesis in resident microglial cells, which in the ischemic lesions are supplemented by TNF synthesizing, blood-borne macrophages. In situ hybridization for TNF mRNA is performed using alkaline phosphatase-labelled oligodeoxynucleotide probes. These probes show excellent rendition of individual cells, and can successfully be combined with immunohistochemical procedures. We also describe a sensitive immunohistochemical method for detection of TNF, which can be combined with visualization of an additional antigen. The specificity of the histological procedures are confirmed by RT-PCR and Western blot analysis on homogenates prepared from microdissected brain regions. Advantages and disadvantages of the methods are discussed with emphasis on the specificity and sensitivity of the histological procedures. Our strategy for detection of TNF mRNA and protein provides a solid basis for clarifying the cellular synthesis, regulation and function of TNF in the normal, injured or diseased CNS. Furthermore, the methodology can readily be applied in studies of other cytokines and growth factors in the CNS.
KW - Animals
KW - Blotting, Western
KW - Cerebral Cortex
KW - DNA Primers
KW - Female
KW - Glial Fibrillary Acidic Protein
KW - Immunohistochemistry
KW - In Situ Hybridization
KW - Infarction, Middle Cerebral Artery
KW - Macrophage-1 Antigen
KW - Male
KW - Mice
KW - Mice, Inbred Strains
KW - Microglia
KW - Perforant Pathway
KW - RNA, Messenger
KW - Reverse Transcriptase Polymerase Chain Reaction
KW - Sensitivity and Specificity
KW - Tumor Necrosis Factor-alpha
U2 - 10.1016/s1385-299x(01)00062-9
DO - 10.1016/s1385-299x(01)00062-9
M3 - Journal article
C2 - 11356385
SN - 1385-299X
VL - 7
SP - 175
EP - 191
JO - Brain Research Protocols
JF - Brain Research Protocols
IS - 2
ER -