α bungarotoxin alexa fluor 488 conjugate

Cryosections were fixed in 4% formaldehyde (Sigma-Aldrich, Saint Louis, Missouri, #F8775) for 10 min at room temperature, then washed and blocked with 1% BSA (Sigma-Aldrich, Saint Louis, Missouri, #A9647) and 0.2% Triton (Sigma-Aldrich, Saint Louis, Missouri, # X100) for 1 h. Samples were then incubated with a 1:100 dilution in 1% BSA of rabbit polyclonal anti-Neurofilament antibody (Genetex, Hsinchu City, Taiwan, # GTX110065) or 1:200 anti-p62/SQSTM1 (C terminus) (Progen, #GP62-C) overnight at 4 °C, followed by incubation with a 1:500 dilution in 1% BSA of Alexa Fluor 568 anti-rabbit (ThermoFisher, Waltham, Massachusetts, #A11011) and α-bungarotoxin Alexa fluor-488 conjugate (ThermoFisher, Waltham, Massachusetts, # B13422), or Alexa Fluor 488 anti-guinea pig IgG (ThermoFisher, Waltham, Massachusetts, #A11073) for one hour at room temperature. Nuclei were stained with TO-PRO-3 iodide (ThermoFisher, Waltham, Massachusetts, #T3605).