Brain stains: optimization of immunohistochemical methods for cleared thick neural tissue

Abstract

Populations of GABAergic interneurons in the prefrontal cortex have been implicated in control and coordination of complex behaviors such as motivation and decision-making. Although the functions of each of the main GABAergic subtypes, SST, PV, and VIP, have been extensively investigated independently, a lack of techniques allowing simultaneous recording has precluded the ability to characterize the interactions between these cell types. Staining for markers of GABA cell types in thick tissue, and relating identities back to in vivo recordings, will allow explicit and direct characterization of how activity covaries in these populations. However, there has been minimal success when staining for GABAergic subtypes in thick tissue. I interrogated the different factors that might be affecting successful thick tissue staining using the Cuboid Method which enables controlled and efficient thick tissue preparation, staining, and experimental testing. Fixation, with both paraformaldehyde and glutaraldehyde, was observed to be a major factor influencing the ability of different SST, PV, and VIP antibodies to label their epitopes in thick tissue. Additionally, fixation appeared to affect SST and VIP staining differently than PV staining. SDS treatment prior to staining was shown to be compatible with GABAergic subtype staining while SDS treatment after staining was shown to dramatically decrease staining signal. Like others’ findings, blocking was shown to be unnecessary for better PV and SST staining. Lastly, relative proportions of SST, PV, and VIP in the PFC and their staining patterns in thick tissue matched results from previous work in thin tissue to support that staining was successful and also accurate. This research provides the knowledge necessary to successfully stain for SST, PV, and VIP in thick tissue in addition to showing the efficacy of the Cuboid Method for testing factors affecting staining in thick tissue.