Meeting Abstract
Honeybees have been used as a model organism for learning, behavior and memory. Brain imaging techniques are fundamental for understanding how the brain works. Immunofluorescence labeling combined with confocal microscopy is a powerful approach to understand behavior from a molecular standpoint. However, this technique is associated with signal-noise issues related to autofluorescence, fixation and light penetrance. In this study, we sought to optimize conditions for staining whole mount Honeybee brains by decreasing fixation-related autofluorescence using NaBH4 and increase light penetrance using brain clearing methods. Bee heads were fixed in formaldehyde and stored in a glycol-based antifreeze to preserve the tissue, then brains were incubated with NaBH4. Immunohistochemical assays were conducted for pigment dispensing factor-PDF. Focus Clear™, a reagent that increases tissue transparency was used before mounting. Results show that autofluorescence is reduced when combining NaBH4 with Triton, a detergent that increases tissue permeability. One-way ANOVA analysis on average fluorescence intensity shows significant differences between the untreated and treated samples. Images taken from the confocal microscope show a higher amount of optical sections for brains treated with clearing agent due to improved light penetrance on the tissue. Brains incubated with anti-PDF did not exhibit immunoreactivity. All data shows the techniques used helped reduce autofluorescence and increase light penetration.
