Laser microdissection of immunolabeled astrocytes allows quantification of astrocytic gene expression.
Astrocytes represent the major glial cell population within the central nervous system. In order to elucidate the function of astrocytes under physiological conditions and during the course of neurological disease, astrocytic gene expression profiling is necessary. However, since astrocytes form an intimately connected network with neurons and other cell types in the brain, gene expression analysis of astrocytes with a sufficient degree of cellular specificity is difficult. Here we are presenting a rapid and, thus, RNA preserving immunostaining protocol for the detection of astrocytes in rodent brain. This protocol can readily be combined with laser microdissection (Leica AS LMD platform) and quantitative RT-PCR (qPCR). Employing this method, we studied changes in glial fibrillary acidic protein (GFAP) expression in astrocytes of mouse entorhinal cortex following entorhinal cortex lesion. Using laser microdissection, astrocytes (n = 60) were collected in the tissue surrounding the lesion, the entorhinal cortex contralateral to the lesion, and in unlesioned control animals. Changes in GFAP mRNA were quantified using qPCR. GFAP mRNA levels were 82-fold higher in astrocytes of lesioned animals at the site of the lesion compared to GFAP mRNA levels in entorhinal cortex astrocytes of control mice. GFAP mRNA levels were only slightly elevated at the contralateral side (lesioned animals). This optimized protocol for immunolabeling and laser microdissection of astrocytes followed by qPCR allows quantification of astrocytic gene expression levels with a high degree of cellular specificity. It may similarly be employed in different settings where other cell types need to be identified and microdissected for gene expression profiling.
Institute of Clinical Neuroanatomy, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
Guido J
GJ
Burbach
Doris
D
Dehn
Barbara
B
Nagel
Domenico
D
Del Turco
Thomas
T
Deller
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D007839:Functional Laterality; D015870:Gene Expression; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods; D007834:Lasers; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D042282:Microdissection / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis; D012337:RNA, Ribosomal, 18S / Q000032:analysis; D012339:RNA, Ribosomal, 28S / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D018709:Statistics, Nonparametric; D015854:Up-Regulation
D005904:Glial Fibrillary Acidic Protein; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D012339:RNA, Ribosomal, 28S