Immunohistochemistry.

Brain tissue was embedded in OCT (Tissue-Tek) and frozen. Coronal sections (18 μm) were cut and mounted onto slides using the CryoJane system (Leica Biosystems). eGFP expression in slices from SynRosa26 mice was directly imaged without use of antibodies. Immunohistochemistry was performed as previously described.²⁸ Sections undergoing ASIC1A immunohistochemistry were post-fixed for 10 minutes in PBS with 4% paraformaldehyde and 4% sucrose, followed by three 5 minutes washes with PBS. Sections were incubated with 0.25% Triton X-100 for 5 minutes, followed by an additional three 5 minutes washes with PBS. Sections were blocked with 5% goat serum for 1 hour and then incubated overnight at 4°C with primary antibodies diluted in 5% goat serum. Rat polyclonal anti-ASIC1A antiserum (MTY19)²⁸ diluted 1:100 was used to identify ASIC1A, a rat monoclonal CD31 antibody (BD Pharmingen, Cat# 550274) diluted 1:500 was used to identify blood vessels, a mouse monoclonal NeuN antibody (Millipore, Cat# MAB377) diluted 1:250 was used to identify neuronal nuclei, and a mouse monoclonal GFAP antibody (Millipore, Cat# MAB360) diluted 1:500 was used to identify astrocytes. After incubation in the primary antibody sections were then washed three times for 5 minutes in PBS, followed by a 1 hour incubation with the secondary antibody. For ASIC1A: goat-anti rabbit IgG coupled to Alexa Fluor 488 (Invitrogen, Cat#A11070), for CD31: goat-anti rat IgG coupled to Alexa Fluor 568 (Invitrogen, Cat#A11077), and for NeuN and GFAP: goat-anti mouse IgG coupled to Alexa Fluor 568 (Invitrogen, Cat#A11019). All secondary antibodies were diluted 1:500 in 5% goat serum. After antibody incubation, slides were washed three times for 5 minutes in PBS and mounted with Vectashield (Vector Labs). Sections were imaged at 10x or 63x with a confocal microscope (Zeiss 710). To control for specificity of the immunofluorescent signals, sections were run in parallel that were exposed to only the primary antibody or only the secondary antibody, and no fluorescence above background levels was observed. To control for the specificity of eGFP signal, a Cre- littermate of the SynRosa26 mice was imaged and no fluorescence above background levels was observed. Blinding was not performed in these experiments, but all punches or slices were treated equally.