DYNtdTOM mice were sacrificed and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The lumbar enlargement of the spinal cord was removed and placed in 30% sucrose (in 0.1M PB) overnight. The tissue was frozen and 40 μm sections were cut using a cryostat (Leica; Wetzlar, Germany). Free-floating sections were washed in phosphate-buffered saline (PBS) and placed in a blocking buffer containing 10% normal donkey serum and 0.3% Triton X-100. The sections were then incubated in a primary antibody against the transcription factor Pax2 (1:500 dilution; ThermoFisher #716000; RRID: AB_2533990) overnight at 4°C. Pax2 was used as a marker of inhibitory neurons since it is continuously expressed throughout development and is present in both GABAergic and glycinergic neurons [39 (link); 52 (link); 62 (link)]. Sections were washed again in PBS and incubated in a species-specific secondary antibody conjugated to AlexaFluor 488 (1:500 dilution; ThermoFisher #A-11008; RRID: AB_143165) for one hour at room temperature.
The spinal cord dorsal horn of each section was imaged on a BX63 upright fluorescent microscope (Olympus; Center Valley, PA) at 20X or 40X, and z-stack images were taken with a separation of 1 μm. The total number of both tdTOM- and tdTOM/Pax2-expressing cells in a section were counted using CellSens Dimension Desktop software (Olympus).
The spinal cord dorsal horn of each section was imaged on a BX63 upright fluorescent microscope (Olympus; Center Valley, PA) at 20X or 40X, and z-stack images were taken with a separation of 1 μm. The total number of both tdTOM- and tdTOM/Pax2-expressing cells in a section were counted using CellSens Dimension Desktop software (Olympus).
