A specialized protocol to minimize lipid and aldehyde auto-fluorescence was utilized (Kupferschmidt et al. 2015 (link)) for histochemistry and immunohistochemistry of 250 μm brain slices used for whole-cell electrophysiology. This protocol was used to illustrate expression of ACC terminals in claustrum and claustrum neurons targeted for whole-cell recordings. For these slices, primary rabbit anti-mCherry antibody (1:500; Abcam, Cambridge, MA) and secondary donkey anti-rabbit antibody conjugated to Cy3 (1:500; Jackson ImmunoResearch, West Grove, PA) were used to label viral expression of mCherry protein. Streptavidin conjugated to AlexaFluor®-488 (1:1,000; Jackson ImmunoResearch) was used to label neurobiotin-filled neurons.
To obtain representative images of retrograde tract tracer injections, mice injected with retrograde BDA were transcardially perfused with room temperature 0.1M phosphate-buffered saline (PBS), pH 7.2-7.4, followed by ice-cold 4% (weight/volume) paraformaldehyde in PBS. After the brain was extracted, brains were post-fixed with 4% paraformaldehyde in PBS overnight at 4°C. Coronal sections were sliced using an Integraslice 7550 MM vibrating microtome (Campden Instruments, Loughborough, England) at a thickness of 50 μm. Sections were immediately mounted and imaged to visualize retrograde BDA fluorescence.
To obtain representative images of retrograde tract tracer injections, mice injected with retrograde BDA were transcardially perfused with room temperature 0.1M phosphate-buffered saline (PBS), pH 7.2-7.4, followed by ice-cold 4% (weight/volume) paraformaldehyde in PBS. After the brain was extracted, brains were post-fixed with 4% paraformaldehyde in PBS overnight at 4°C. Coronal sections were sliced using an Integraslice 7550 MM vibrating microtome (Campden Instruments, Loughborough, England) at a thickness of 50 μm. Sections were immediately mounted and imaged to visualize retrograde BDA fluorescence.
