TY - JOUR
T1 - A simplified method for combined immunohistochemistry and in-situ hybridization in fresh-frozen, cryocut mouse brain sections
AU - Newton, Sathyanesan Samuel
AU - Dow, Antonia
AU - Terwilliger, Rose
AU - Duman, Ronald
PY - 2002
Y1 - 2002
N2 - A method is described to perform combined immunohistochemistry and in situ hybridization in mouse brain sections. The protocol is specific to sections mounted on glass slides. In contrast to earlier methods that require either paraffin embedding or perfusion of the brain with paraformaldehyde, this protocol can be carried out on fresh-frozen, cryostat cut post-fixed sections. This simple and concise protocol increases the applicability of the technique as the RNAse-free immunodetection of antigen is useful by itself for immunologically identifying specific cells of interest and then examining gene expression in those cells using techniques such as real-time PCR and microarray analysis. The use of fresh-frozen, cryocut sections enables reliable detection of easily perturbable post-translational modifications such as phosphorylation and improves the quality of results obtained in subsequent in situ hybridization by reducing the background signal and interference from lower cell layers. Inducible transgenic mice that express either a dominant negative mutant form of the cAMP response element binding protein (mCREB) or CREB, in discrete brain regions, were used in this study. The combined immunohistochemistry and in situ hybridization protocol was used to examine colocalization of enkephalin or dynorphin mRNA, both downstream targets of CREB-mediated gene expression, in cells expressing transgenic mCREB or CREB.
AB - A method is described to perform combined immunohistochemistry and in situ hybridization in mouse brain sections. The protocol is specific to sections mounted on glass slides. In contrast to earlier methods that require either paraffin embedding or perfusion of the brain with paraformaldehyde, this protocol can be carried out on fresh-frozen, cryostat cut post-fixed sections. This simple and concise protocol increases the applicability of the technique as the RNAse-free immunodetection of antigen is useful by itself for immunologically identifying specific cells of interest and then examining gene expression in those cells using techniques such as real-time PCR and microarray analysis. The use of fresh-frozen, cryocut sections enables reliable detection of easily perturbable post-translational modifications such as phosphorylation and improves the quality of results obtained in subsequent in situ hybridization by reducing the background signal and interference from lower cell layers. Inducible transgenic mice that express either a dominant negative mutant form of the cAMP response element binding protein (mCREB) or CREB, in discrete brain regions, were used in this study. The combined immunohistochemistry and in situ hybridization protocol was used to examine colocalization of enkephalin or dynorphin mRNA, both downstream targets of CREB-mediated gene expression, in cells expressing transgenic mCREB or CREB.
KW - Colocalization
KW - Combined immunohistochemistry and in situ hybridization
KW - Gene expression
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UR - http://www.scopus.com/inward/citedby.url?scp=0036310695&partnerID=8YFLogxK
U2 - 10.1016/S1385-299X(02)00148-4
DO - 10.1016/S1385-299X(02)00148-4
M3 - Article
C2 - 12113781
AN - SCOPUS:0036310695
SN - 1385-299X
VL - 9
SP - 214
EP - 219
JO - Brain Research Protocols
JF - Brain Research Protocols
IS - 3
ER -