Brain sections were co-stained for the expression of the oxidative stress
marker nitrotyrosine and the neuron-specific neuronal nuclear antigen
(NeuN), nitrotyrosine, and the astrocyte marker the glial fibrillary acidic
protein (GFAP) or the apoptotic marker cleaved caspase-3 and NeuN as
previously described.34 (link),35 (link),38 (link) Briefly,
free-floating sections were rinsed in phosphate-buffered saline (PBS) and
blocked in 1% horse serum in PBS containing .3% Triton X for 1 h. Sections
were then transferred in an antibody mixture containing rabbit
anti-nitrotyrosine polyclonal antibody (1:2500; Sigma, MO) and chicken
anti-GFAP polyclonal antibody (1:3000; Novus Biologicals, CO), rabbit
anti-nitrotyrosine polyclonal antibody (1:2500; Sigma, MO) and mouse
anti-NeuN monoclonal antibody clone, A60 (1:1500; Millipore/Sigma, MO) or
rabbit anti-cleaved caspase 3 polyclonal antibody (1:7500; Millipore, MA)
and mouse anti-NeuN monoclonal antibody clone, A60 (1:1500 Millipore/Sigma,
MO) and incubated at 4°C overnight. Sections were washed in PBS and
incubated in a mixture of corresponding secondary antibodies containing
Alexa Fluor 594 and Alexa Fluor 488 or Alexa Fluor 547 (1:2000; Invitrogen,
NY) for 1 h at room temperature. Sections were washed with PBS,
counterstained with 4,6-diamidino-2-phenylindole (DAPI), and mounted with an
anti-fade mounting medium (Vector Labs, CA).
marker nitrotyrosine and the neuron-specific neuronal nuclear antigen
(NeuN), nitrotyrosine, and the astrocyte marker the glial fibrillary acidic
protein (GFAP) or the apoptotic marker cleaved caspase-3 and NeuN as
previously described.34 (link),35 (link),38 (link) Briefly,
free-floating sections were rinsed in phosphate-buffered saline (PBS) and
blocked in 1% horse serum in PBS containing .3% Triton X for 1 h. Sections
were then transferred in an antibody mixture containing rabbit
anti-nitrotyrosine polyclonal antibody (1:2500; Sigma, MO) and chicken
anti-GFAP polyclonal antibody (1:3000; Novus Biologicals, CO), rabbit
anti-nitrotyrosine polyclonal antibody (1:2500; Sigma, MO) and mouse
anti-NeuN monoclonal antibody clone, A60 (1:1500; Millipore/Sigma, MO) or
rabbit anti-cleaved caspase 3 polyclonal antibody (1:7500; Millipore, MA)
and mouse anti-NeuN monoclonal antibody clone, A60 (1:1500 Millipore/Sigma,
MO) and incubated at 4°C overnight. Sections were washed in PBS and
incubated in a mixture of corresponding secondary antibodies containing
Alexa Fluor 594 and Alexa Fluor 488 or Alexa Fluor 547 (1:2000; Invitrogen,
NY) for 1 h at room temperature. Sections were washed with PBS,
counterstained with 4,6-diamidino-2-phenylindole (DAPI), and mounted with an
anti-fade mounting medium (Vector Labs, CA).
