Procedure
- Isotropic Fractionation
The procedure of isotropic fractionation consists of transforming the brain in a suspension of free cell nuclei where total number of cell and neurons can be estimated thereafter through fluorescent staining.
2. Dapi Staining and Cell Counting
DAPI staining is a technique used to stain cells or tissues, as it penetrates the cell membrane and binds to the DNA present in the nucleus. This allows to visualize the nuclei of cells under a fluorescence microscope and count the number of cells.
3. NeuN Staining and Neuron Counting
NeuN staining is a technique used ito identify neurons in tissue samples, as it relies on an antibody that specifically binds to a protein called neuronal nuclear antigen (NeuN), which is predominantly expressed in the nuclei of neurons in the central nervous system.
This allows to discriminate which cells in our samples are neurons, and thus count the number of neurons and non neurons.
4. Statistical Analysis
The effect of treatment on brain cell numbers were investigated using Linear Regression Analysis.
The potential influence of sex and allometric variable were taken in account. Furthermore, when analysing for Treatment effects, the dataset was restricted only to the 54 individuals hatched from eggs which were laid 4 days after cort-implantation due to the reported influence of this condition on maternal stress treatment effects.