Mouse anti neuronal nuclei neun

The following antibodies were used: rabbit anti-ATF3 (1:200, Santa Cruz Biotechnology Inc.), mouse anti-neurofilament 200 (NF200; clone N52, 1:500, Sigma), rabbit anti-calcitonin gene related peptide (CGRP; 1:4000, Sigma), rabbit anti-tyrosine hydroxylase (TH; 1:1000, Millipore). To identify non-peptidergic nociceptors, slides were first incubated in isolectin B4 (IB4;1:400, Sigma) followed by an anti-IB4 primary antibody (1:2000, Vector laboratories). To characterize recombined neurons in the brain we used mouse anti-Neuronal Nuclei (NeuN; 1:100, Millipore) and rabbit anti-calbindin d28k (1:1000, Swant). Secondary antibodies included donkey anti-rabbit Alexa-488, donkey anti-goat Alexa-647, donkey anti-mouse Dylight 650 (all at 1:1000, Invitrogen), donkey anti-mouse aminomethylcoumarin (AMCA; 1:100, Jackson labs). Nuclear counterstains were 4′,6-diamidino-2-phenylindole (DAPI) (in ProLong Gold coverslipping medium, Invitrogen) or Hoechst 33342 (1:10,000, Sigma).

Immunostaining was performed as described above. The following primary antibodies were used: rabbit polyclonal anti-doublecortin (anti-DCX, 2 μg/mL) (Cell Signaling Technology, Danvers, MA, USA), mouse anti-glial fibrillary acid protein (anti-GFAP, 2 μg/mL) (Millipore), mouse anti-neuronal nuclei (NeuN, 2 μg/mL) (Millipore), rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (anti-Iba1, 2 μg/mL) (Wako Pure Chemical Industries, Osaka, Japan), and mouse monoclonal anti-macrophages/monocytes clone ED1 (anti-ED1/CD68, 2 μg/mL) (Millipore). The following secondary antibodies (4 μg/mL) were subsequently applied: goat Alexa 594-conjugated anti-rat IgG, goat Alexa 594-conjugated anti-mouse IgG, goat Alexa 594-conjugated anti-mouse IgM, and goat Alexa 594-conjugated anti-rabbit IgG (Invitrogen). Sections were examined with an LSM510 or LSM610 confocal microscope (Carl Zeiss).