Objective

2

To establish a quantitative detection system for phosphorylated protein kinase A (p-PKA) in labeled hippocampal neurons of mice following mild brain trauma using laser microdissection combined with automated capillary electrophoresis Western blotting.

Methods

2

From a mouse model of mild traumatic brain injury

fresh samples of the hippocampal tissues were collected for preparing frozen sections with Thionine staining. Laser microdissection was used for collecting the neurons in the hippocampal CA1 and DG regions and the expression level of p-PKA was detected using fully automated capillary Western blotting.

Results

2

The lower detection limit of automated capillary electrophoresis Western blotting for low-concentration proteins was lower than that of conventional Western blotting by 1 order of magnitude. This combined detection system had a linear correlation coefficient of 98.85% for p-PKA detection with inter- and intra-day precisions (RSD) of 2.50% and 4.09%

respectively. In the mouse model with traumatic brain injury

the level of p-PKA in the hippocampal DG region increased significantly at 1 day after the injury (

P

<

0.01)

but its expression showed no significant changes in the CA1 region.

Conclusions

2

Laser microdissection combined with automatic capillary Western blotting allows collection of specific neuronal cells and accurate quantification of low-concentration proteins

and can be potentially used for cellular and molecular studies of tissues with complex distribution of different cell types.