Retinae and optic nerves were collected immediately after the mice were euthanized, snap frozen and placed on dry ice, and stored at −80 °C in a freezer. NP-40 extracts were prepared with Bullet blender (Next Advance, BBX24). Free fatty acids (FFA) were measured with the FFA Quantification kit as described [34 (link),55 (link)] and as per manufacturer’s instructions (Biovision, Mountain View, CA). A colorimetric assay kit for acetylcholinesterase activity was used as directed per manufacturer (Biovision) and previously described [34 (link)]. Data were normalized against protein amounts in NP40-solubilized tissue extracts used for each assay. Protein concentrations of NP40-solubilized extracts were determined by the Bradford assay (BioRad).
Protocol detail
Quantifying Free Fatty Acids and Acetylcholinesterase in Mouse Retinae and Optic Nerves
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Acetylcholinesterase
Assay
Colorimetric
Dry ice
Free fatty acids
Frozen
Mice
Np 40
Optic nerves
Protein
Retinae
Tissue extracts
Corresponding Organization : Duke Medical Center
Other organizations : Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland Eye Clinic, Cleveland Clinic, Louis Stokes Cleveland VA Medical Center
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Variable analysis
independent variables
- None explicitly mentioned
- Free fatty acids (FFA) measured with FFA Quantification kit
- Acetylcholinesterase activity measured with colorimetric assay kit
- Retinae and optic nerves collected immediately after mice were euthanized
- Retinae and optic nerves snap frozen and placed on dry ice
- Retinae and optic nerves stored at -80°C in a freezer
- NP-40 extracts prepared with Bullet blender
- Protein concentrations of NP40-solubilized extracts determined by Bradford assay
- Positive control: Not specified
- Negative control: Not specified
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