Mice were given a lethal dose of anesthesia (Ketamine 100 mg/kg, Xylazine 10 mg/kg) and perfused transcardially with 0.9% NaCl followed by 4% para formaldehyde (PFA). Mouse brains were post fixed in 4% PFA overnight and transferred to a 30% sucrose solution for 48 hours. Coronal sections of 40 µm thickness were prepared with a sliding microtome. Brain sections of one-in-six series were selected for immunostaining (for BrdU cell counting, rat anti-BrdU, 1:100, Acurate) or DAPI staining. The characterization of rat anti-BrdU has been previously documented (Mathews et al., 2010 (link)). BrdU+ or GFP+ cells in the GC layer were visualized and counted with a Nikon E800 microscope (Melville, NY). The total number of labeled cells per DG was estimated by multiplying the number counted from the one-in-six series by 6.
Protocol detail
Quantifying BrdU-labeled Neurons in Mouse Dentate Gyrus
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0 9 nacl
Anesthesia
Brain
Brdu
Cells
Dapi
Formaldehyde
Ketamine
Microscope
Microtome
Mouse
Sucrose
Xylazine
Corresponding Organization :
Other organizations : Salk Institute for Biological Studies
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Variable analysis
independent variables
- Lethal dose of anesthesia (Ketamine 100 mg/kg, Xylazine 10 mg/kg)
- Number of BrdU+ or GFP+ cells in the GC layer of the dentate gyrus
- Perfusion with 0.9% NaCl followed by 4% paraformaldehyde (PFA)
- Post-fixation of mouse brains in 4% PFA overnight
- Transfer of mouse brains to 30% sucrose solution for 48 hours
- Preparation of 40 µm thick coronal brain sections using a sliding microtome
- Selection of brain sections from one-in-six series for immunostaining or DAPI staining
- Positive control: Characterization of rat anti-BrdU antibody (Mathews et al., 2010)
- Negative control: Not explicitly mentioned
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