Immunohistochemistry was performed on formalin-fixed sections, according to[9 (link)]. Brains were perfused transcardially first with ice cold PBS, then with 4% formalin. Following rapid removal and overnight storage in 4% formalin, brains were transferred to buffer containing 15% sucrose for 24 hours, then 30% sucrose. 20 μm sections are cut on a freezing microtome. After staining with primary antibodies (an affinity-purified antiserum raised against the C-terminus of 12/15-LOX in rabbits[13 (link)], antibodies to AIF (D-20, Santa Cruz Biotechnology, Dallas, TX), activated caspase-3 (Asp-175, Cell Signaling, Danvers, MA), and fluorescent-tagged secondary antibodies, sections were analyzed on a Nikon Eclipse Ti fluorescent microscope equipped with NIS Elements software. For co-localization studies, a Zeiss LSM 5 Pascal scanning confocal microscope was used.
Protocol detail
Immunohistochemical Analysis of Brain Tissue
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15 lox
Antibodies
Antiserum
Brains
Buffer
Caspase 3
Cold
Confocal microscope
Fluorescent antibodies
Formalin
Immunohistochemistry
Microscope
Microtome
Rabbits
Sucrose
Corresponding Organization :
Other organizations : Massachusetts General Hospital, Harvard University
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Variable analysis
independent variables
- Primary antibodies used (an affinity-purified antiserum raised against the C-terminus of 12/15-LOX in rabbits, antibodies to AIF (D-20, Santa Cruz Biotechnology, Dallas, TX), activated caspase-3 (Asp-175, Cell Signaling, Danvers, MA))
- Immunohistochemistry results (analyzed on a Nikon Eclipse Ti fluorescent microscope equipped with NIS Elements software)
- Co-localization results (analyzed on a Zeiss LSM 5 Pascal scanning confocal microscope)
- Formalin-fixed sections
- Perfusion of brains with ice cold PBS and 4% formalin
- Overnight storage in 4% formalin, followed by transfer to buffer containing 15% and 30% sucrose
- 20 μm sections cut on a freezing microtome
- Fluorescent-tagged secondary antibodies
- Not explicitly mentioned
- Not explicitly mentioned
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