After fixation, the cassette containing the gel block was passed through a circulating machine (STP 120 Thermo Scientific) for paraffin impregnation. We tried to use the same program that Nam et al. used but with some changes to fit our machine [10 (link)].
Their program was 13 h under vacuum impregnation at 20 lbs; the vacuum allowed faster penetration of the fluids. Our machine was not equipped with a vacuum device, so we made changes in terms of increasing the bath times. The final program was as follows: 80% ethanol (AnalaR NORMAPUR VWR) with one change (1.15 h each), 96% ethanol (1 h), 100% ethanol with 2 changes (2 h each), ethanol/toluene 1 : 1 (1 h); toluene with 3 changes (1 h each), and paraffin wax at 60°C (2 h), due to the absence of vacuum in our apparatus.
Their program was 13 h under vacuum impregnation at 20 lbs; the vacuum allowed faster penetration of the fluids. Our machine was not equipped with a vacuum device, so we made changes in terms of increasing the bath times. The final program was as follows: 80% ethanol (AnalaR NORMAPUR VWR) with one change (1.15 h each), 96% ethanol (1 h), 100% ethanol with 2 changes (2 h each), ethanol/toluene 1 : 1 (1 h); toluene with 3 changes (1 h each), and paraffin wax at 60°C (2 h), due to the absence of vacuum in our apparatus.
