The engineering of genetically modified mice which express fluorescent proteins only in astrocytes was an important step for reliable identification of astrocytes. Nimmerjahn et al. (2004) described an alternative method for selective in vivo staining of astroglia in the neocortex using sulforhodamine 101 (SR101). This method was adapted by other groups for in vitro stainings in the hippocampus. Our aim was to adapt this method for the brainstem. Using acute brainstem and hippocampus slices of TgN(hGFAP-EGFP) mice, we performed SR101 stainings according to the protocol of Langer & Rose (2009). After staining, we imaged 40 mm stacks using the 2-photon setup. For analysis, the number of astrocytes per stack was counted and fluorescence intensity was measured for EGFP and SR101. We used EGFP fluorescence to reliable identify and count the astrocytes per stack. Using hippocampus slices as control, we found that SR101 staining in the brainstem was very weak. While the number of stained cells was comparable to the hippocampus, intensity was too weak for identification of astrocytes. After blocking gap-junction hemichannels with carbenoxolone, SR101 staining was almost completely blocked in hippocampus slices. In contrast to that, pannexin blockade with mefloquine did not affect staining efficacy. Immunostainings against pannexin1 revealed astroglial pannexin1 expression in brainstem and hippocampus, but we found no differences of pannexin1 expression in these two regions. While we could confirm selective astroglial staining of SR101 in the hippocampus, this method is not applicable for identification of astrocytes in the brainstem. Furthermore our data excludes that the labeling differences in hippocampus and brainstem are due to different pannexin1 expression.