A method to perform Western blots of microscopic areas of histological sections.
Western blotting is one powerful research method to specifically detect proteins. However, it has been barely possible to investigate microscopic volumes of tissue so far because of the required minimum volumes and the pretreatment. Herein, we describe a method of performing Western blots directly from the histological section of frozen or paraffin-embedded tissue. Small histological areas of a mouse brain were lysed by section lysis buffer, subjected to a miniaturized SDS-PAGE, and detected by immunoblotting. Thereby, an area equivalent to only 15 cortical neurons of mouse cortex was detectable. This offers the possibility of correlating histological findings to biochemical investigations. In addition, enzymatic pretreatment was applied to identify the glycosylation of the major cleavage product of the prion protein. Moreover, the section lysis buffer is a sophisticated method to conserve and investigate phosphorylation sites as demonstrated here by phopsphorylated Akt and ERK. The presented technique combines histology with Western blotting techniques and will be of value for investigations of discrete tissue areas.
Krebs
Bjarne
B
Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377, Munich, Germany.
Kohlmannsperger
Veronika
V
Nölting
Svenja
S
Schmalzbauer
Rüdiger
R
Kretzschmar
Hans A
HA
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005629:Frozen Sections; D006031:Glycosylation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008832:Microchemistry; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D016612:Paraffin Embedding; D010766:Phosphorylation; D011328:Prions / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism
D011328:Prions; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3