The procedure was performed in the hippocampal CA3 field from postnatal day 53 (P53) rat brain as described previously (Wilczynski et al., 2008 (link)). For immunodetection, sheep polyclonal anti-CD44 (1:50; R&D Systems, Minneapolis, MN) followed by secondary antibodies coupled to 10-nm gold particles was applied (Electron Microscopy Sciences, Fort Washington, PA). Nonimmune sheep IgG (BD PharMingen, San Jose, CA) was used as a negative control. Gold particle densities within various ultrastructural compartments were measured on digital micrographs using ImageJ software (National Institutes of Health, Bethesda, MD). The statistical evaluation of the labeling was performed using χ2 test of observed and expected gold counts over the given compartments (Mayhew and Lucocq, 2008 (link)). The data were sampled randomly. At least 200 gold particles at ∼10 images from three animals per group were counted.
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CD44 Protein Immunodetection in Hippocampal CA3
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Corresponding Organization : Instytut Biologii Doświadczalnej im. Marcelego Nenckiego
Other organizations : Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Wroclaw Medical University
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Variable analysis
independent variables
- Postnatal day (P53) of the rat brain
- Gold particle densities within various ultrastructural compartments
- Hippocampal CA3 field of the rat brain
- Immunodetection using sheep polyclonal anti-CD44 antibody (1:50; R&D Systems, Minneapolis, MN)
- Nonimmune sheep IgG (BD PharMingen, San Jose, CA) as a negative control
- Positive control: Sheep polyclonal anti-CD44 antibody
- Negative control: Nonimmune sheep IgG
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