Dibutylphthalate polystyrene xylene (dpx)

Immunohistochemistry was conducted on the tissue to enhance the fluorescence signal of mCherry (DREADD groups) or EGFP (control groups). The first series of sections was transferred from PBS into a blocking solution of 5% NGS in PBS with Triton X-1000 (PBST) and incubated for 1 h. The sections were then transferred into the primary antibody solution of rabbit-anti-mCherry or chicken polyclonal anti-GFP (Abcam) at a dilution of 1:1000 in PBST with 1% NGS and incubated for 24 h. Sections were washed 4 times in PBST and transferred to a secondary antibody solution of goat-anti-rabbit (Dylight AlexaFlour-594, Vector Laboratories) or goat-anti-chicken (Invitrogen) at a dilution of 1:200 at PBST. From this point onward, the sections were kept in the dark. Sections were incubated for 1 h before being washed 3 times in PBS. Sections were mounted onto gelatin-subbed glass slides and dried overnight before being immersed in xylene and coverslipped using DPX (Thermo Fisher Scientific). All incubations were on a stirring plate at room temperature, and all washes were for 10 min.

FJB stains all degenerating neurons regardless of specific insult or mechanism of cell death. FJB staining was performed, as described previously [37 (link)]. In short, the sections were soaked in 0.06% potassium permanganate and incubated in 0.0004% FJB (Cell Signaling Technology, Beverly, MA, USA). Thereafter, these sections were washed and put on a hot plate (about 50 °C) for the reaction of FJB. Finally, the sections were cleared in xylene and coverslipped with DPX (Thermo Fisher Scientific, Waltham, MA, USA).

Immunohistochemistry was carried out on the tissue to enhance the fluorescence signal of mCherry (DREADD groups). The first series of sections was transferred from PBS into a blocking solution of 5% normal goat serum (NGS) in PBS with Tritonx-1000 (PBST) and incubated for 1 h. The sections were then transferred into the primary antibody solution of rabbit-anti-mCherry (Abcam) at a dilution of 1:1000 in PBST with 1% NGS and incubated for 24 h. Sections were washed 4 times in PBST and transferred to a secondary antibody solution of goat-anti-rabbit (Dylight Alexa flour 594, Vector Laboratories) or at a dilution of 1:200 at PBST. From this point onwards the sections were kept in the dark. Sections were incubated for 1 h before being washed 3 times in PBS. Sections were mounted onto gelatine-subbed glass slides and allowed to dry overnight before being immersed in xylene and coverslipped using DPX (Thermo Fisher Scientific). All incubations were on a stirring plate at room temperature and all washes were for 10 min. Virus expression was analyzed using a fluorescent Leica DM5000B microscope with a Leica DFC310 FX camera. Images were collected from the ACC, selected efferent targets, and a comparison cortical area, secondary somatosensory cortex.

DNA fragmentation was evaluated using an ApopTag1 (Qbiogene) kit. In this method, the enzyme terminal deoxynucleotidyl transferase labels the 3-OH ends of DNA generated during apoptosis with biotinylated nucleotides. Immunoperoxidase staining was used to detect these fragments. The apoptosis detection kit enables distinction of apoptosis from necrosis by specifically detecting the DNA cleavage and chromatin condensation associated with apoptosis. Human melanoma cells were cultured on 8-well slides (20,000 cells per slide, Roth Germany) overnight for attachment. Then, the cells were treated with the investigated compounds at concentrations of 6 and 12.5 µM for 24 h. After treatment, the cells were fixed with 4% formalin in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature. TUNEL assay was carried out according the manufacturer’s (Millipore) instructions. Nuclei were stained with hematoxylin. Then samples were mounted by DPX (Thermo Fisher Scientific, Germany) on glass slides. Cells with stained nuclei were investigated by counting 100 cells in 3 randomly selected fields. The analyses were performed by two independent investigators. Samples were evaluated with a BX51 upright light microscope (Olympus, Japan).

Formalin-fixed, paraffin-embedded tissue sections were deparaffinised and rehydrated. Heat-induced antigen retrieval was performed using a 10 mM sodium citrate buffer with 0.05% Tween-20 (pH 6.0) at 95°C for 15–20 min. Hydrogen peroxidase block was performed for 15 min followed by further incubation with blocking buffer for 1 h. Abcam protein block was applied for 10 min and then sections were incubated in primary antibody at 4°C overnight in a wet chamber. Sections were incubated with biotinylated goat anti-polyvalent for 10 min and covered with streptavidin peroxidase for a further 10 min. 3, 3′-diaminobenzidine (DAB) chromogen (HRP/DAB (ABC) Detection Kit, Abcam) was added to DAB substrate, applied to sections, and incubated for 1–3 min. Haematoxylin counterstain was performed, washed in tap water and slides dipped into Scott's Tap Water Substitute. Sections were dehydrated and mounted using DPX (Fischer Scientific). Slides were imaged with an Axio Imager optical microscope (ZEISS).

For haematoxylin & eosin (H&E) staining, sections were deparaffinised in Histoclear and gradually rehydrated in ethanol. Slides were incubated in Mayer's haematoxylin (Sigma‐Aldrich) for 5 min and rinsed in distilled water for 30 s before being incubated with alkaline alcohol for 30 s to ensure the nuclei appear blue. Samples were dehydrated, before being incubated with eosin (Sigma‐Aldrich) for 30 s, and then further dehydrated prior to mounting with DPX (Thermo Fisher Scientific) ready for microscopy using a Leica ICC50 high definition camera mounted onto a Brightfield Leica microscope.

Alvetex^® Scaffold 3D paraffin embedded samples were sectioned at 6 μm using a Leica Microtome RM2125RT and mounted onto charged superfrost microscope slides (ThermoFisher Scientific). Sections were deparaffinised in Histoclear and rehydrated. Slides were incubated in Mayer's haematoxylin (Sigma-Aldrich) for 5 min and rinsed in distilled water for 30 s before being incubated with alkaline alcohol for 30 s to ensure the nuclei appear blue. Samples were then dehydrated, before being incubated with eosin (Sigma-Aldrich) for 1 min and then further dehydrated and incubated in Histoclear for 3 min prior to mounting with DPX (ThermoFisher Scientific) ready for microscopy using a Leica ICC50 high definition camera mounted onto a Leica microscope.