Tissue tek o c t compound

Cryosections were prepared after fixation of the IVDs in 4% formaldehyde solution, immersion in 15% and 30% sucrose followed by cartilaginous endplate removal and embedding in Tissue-Tek^® O.C.T.™ Compound (Sysmex, Horgen, Switzerland). Samples were then frozen in liquid nitrogen and cut into 16 µm transversal sections with a cryotome (microm HM560, Thermo Fisher Scientific, Basel, Switzerland). Poly(methyl methacrylate) (PMMA) embedded samples were dehydrated after fixation prior to their embedding in PMMA, and 6 µm sagittal sections were performed by using a microtome (microtome Leica 2155, Leica, Wetzlar, Germany). Sections were stained with Hematoxylin & Eosin (H&E) and with Safranin-O/Fast Green. Additionally, Picrosirius Red staining was performed on PMMA sections.

To further validate the efficacy of TeTxLC cleavage of VAMP2, the effect on the transcriptome expression of BA-NAc neurons was investigated. Naive mice (n = 12) underwent stereotactic surgery and were allocated to either the experimental group BA TeTxLC × NAc retro-Cre (n = 6, BW = 31.3 ± 1.5 g) or the control group BA TeTxLC × NAc retro-mCherry (n = 6, BW = 33.0 ± 3.2 g). At 15 days post-surgery mice were deeply anaesthetised and perfused with PBS at RT for 1 min. The brain was removed and placed in a cryo-mould (E6032-1CS, Sigma) containing embedding medium (Tissue-TEK OCT Compound, Sysmex). The cryo-mould was then placed on powdered dry ice for 10 min, wrapped in aluminium foil, placed in a polythene bag and stored at −80 °C, prior to laser capture microdissection (see the section, “BA-NAc glutamate neuron population transcriptomics”).