Objective: Non-heme iron staining is very weak in the brain due to its limited iron contents. In order to optimize the iron staining in the brain, different types of fixation solutions were explored and compared in this study. Methods:The brain tissues were cut from mice and fixed with 95% ethanol, or a mixture of methanol: chloroform: acetic acid (volume ratio is 6:3:1), or Bouin's solution or polyformaldehyde. Then, coronal frozen sections were prepared and iron histochemistry was performed to detect the iron staining in cerebral cortex, hippocampus, cerebellum and choroid plexus. Finally, the images were captured and saved for analysis.Results: It was found that the different types of fixation solutions didn't have obvious influences on iron staining when the incubation time was proper in Perl's staining solution. Conclusion:Optimal iron staining could be achieved with a combination of DAB-enhancement, and fixative solution could be selected according to other considerations in experiments. This provided important data for establishing a steady method for positive iron staining in the brain tissues.