68-day-old Protamine-EGFP (PRM1-EGFP) mice (Haueter et al., 2010 (link)) (CD1; B6D2-Tg (Prm1-EGFP)#Ltku/H) were euthanised in a schedule 1 procedure via intraperitoneal injection of sodium pentobarbital followed by decapitation following licensed procedures approved by the Mary Lyon Centre and the Home Office UK. Brains were dissected and cut into four equidistant lateral sections using a scalpel, with region 1 encompassing the olfactory bulb. Regions 2 and 3 were then further sectioned using a vibratome (VT1000S, Leica) set to produce 200 µm sections. Brain sections were kept at 4°C in Hank’s Balanced Salt Solution (HBSS) from death to HPF. A 2 mm biopsy punch was used to excise regions of cortex from lateral slices.
Punches were then placed onto electron microscope grids during freezing. Brain punches on grids were frozen between 3 mm planchettes/carriers (Science Services, Munich, Germany) assembled on mid-plates. Briefly, planchettes were coated with 1% soya-lecithin dissolved in chloroform and the solution allowed to evaporate. This process generates small microvesicles on the surface of the planchette. A flat-sided planchette was placed flat side upwards and a glow discharged (Glocube, Quorum, Lewes, UK) electron microscopy grids (UltraAuFoil, 200 Au mesh, 2/2 Au film; Quantifoil Micro Tools) placed on top; brain punches were then placed onto the grid and submerged in 20% bovine serum albumin (BSA) in HBSS. 3 mm planchettes with a 0.1 or 0.2 mm recess, also coated with 1% soya lecithin, were then placed recess-side-down onto the assembled sandwich and high-pressure frozen in a Leica HPM 100 (Leica Microsystems). The planchette grids were then disassembled under liquid nitrogen, clipped into Autogrids (Thermo Fisher Scientific) and stored at 80 K (liquid nitrogen) for later use.
Punches were then placed onto electron microscope grids during freezing. Brain punches on grids were frozen between 3 mm planchettes/carriers (Science Services, Munich, Germany) assembled on mid-plates. Briefly, planchettes were coated with 1% soya-lecithin dissolved in chloroform and the solution allowed to evaporate. This process generates small microvesicles on the surface of the planchette. A flat-sided planchette was placed flat side upwards and a glow discharged (Glocube, Quorum, Lewes, UK) electron microscopy grids (UltraAuFoil, 200 Au mesh, 2/2 Au film; Quantifoil Micro Tools) placed on top; brain punches were then placed onto the grid and submerged in 20% bovine serum albumin (BSA) in HBSS. 3 mm planchettes with a 0.1 or 0.2 mm recess, also coated with 1% soya lecithin, were then placed recess-side-down onto the assembled sandwich and high-pressure frozen in a Leica HPM 100 (Leica Microsystems). The planchette grids were then disassembled under liquid nitrogen, clipped into Autogrids (Thermo Fisher Scientific) and stored at 80 K (liquid nitrogen) for later use.
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