The effect of post mortem delay, age of the subjects and fixation protocols were examined on immunostaining of the human brain samples.Short post-mortem time (2-5h) is crucial for the quality of human brain samples. Our group uses cerebral cortical samples and hippocampi with this short post-mortem period, which are suitable for fine structural research. Here the advantages and disadvantages of perfusion and immersion fixation were investigated, and the quality of different immunohistochemical stainings (calbindin, calretinin, parvalbumin, nonphosphorylated neurofilament H /SMI32/ among others) was compared in human cortical and hippocampal samples. Blood vessels were examined by collagen type 4.In case of ideal fixation and short post mortem delay, cells showed similar staining with both immersion and perfusion fixation. Calretinin-containing cells showed outstanding sensitivity for post mortem delay and fixation quality. However, considerable number of parvalbumin-positive neurons were preserved in samples with long post mortem delay. After immersion fixation, the walls of blood vessels were occasionally more preserved. We have observed that regions with poor blood circulation, such as the thalamus or amygdala, immersion fixation may give better results. Cerebral small vessel disease caused by atherosclerosis also affects the preservation via sclerotic vessels: in these samples the immersion fixation may result in better and homogeneous fixation. Good quality perfusion fixation resulted in better ultrastructure and immunostaining, as it was observed in the electron microscope.Our results emphasize the considerable influence of post mortem delay and tissue fixation protocol on immunohistochemical experiments.