After the rats were sacrificed, the chest was opened, and the ascending aorta was then infused with ice-cold saline and 4% paraformaldehyde. The T3-T5 DRGs were removed and incubated with 4% paraformaldehyde for 3h at room temperature and then replaced with 30% sucrose for 24h at 4°C. The DRGs were then embedded in Histoprep and were cut at a thickness of 10 μm on a cryostat.
For double immunofluorescence, the DRG sections were incubated with a mixture of anti-ASIC1 (1:1000, abcam, USA), monoclonal neuronal-specific nuclear protein (NeuN) (1:500, Millipore, USA), and anti-ASIC3 (1:1000, abcam, USA) overnight at 4°C. The sections were washed with PBS and then incubated with Alexa 488-conjugated goal anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories, USA) for 2h at room temperature.
For double immunofluorescence, the DRG sections were incubated with a mixture of anti-ASIC1 (1:1000, abcam, USA), monoclonal neuronal-specific nuclear protein (NeuN) (1:500, Millipore, USA), and anti-ASIC3 (1:1000, abcam, USA) overnight at 4°C. The sections were washed with PBS and then incubated with Alexa 488-conjugated goal anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories, USA) for 2h at room temperature.