Mouse anti β3 tubulin

The following antibodies were used: rat anti-BrdU (1:2000; Accurate Chemical and Scientific, Westbury, NY, USA, cat. no. OBT0030G), mouse anti-NeuN (1:500; Millipore, cat. no. MAB377), mouse anti-β3 tubulin (1:5000; Promega, Madison, WI, USA) and mouse anti-GFAP (1:500; Sigma, cat. no. G3893). Sections were incubated overnight at 4°C with a cocktail of the primary antibodies. The rat anti-BrdU was detected with the donkey anti-rat IgG (H+L) labeled with Alexa Fluor® 488 (1:1000; Molecular Probes, Invitrogen, Paisley, UK, cat. no. A21208). The antibodies NeuN, β3-tubulin and GFAP were detected with the donkey anti-mouse IgG (H+L) labeled with Alexa Fluor® 594 (1:1000; Molecular Probes, cat. no. A21203). The sections labeled by double immunofluorescence (BrdU+NeuN, BrdU+β3-tubulin or BrdU+GFAP) were visualized with a confocal laser (spectral) scanning microscope (Leica TCS NT; Leica Microsystems) equipped with a 561 nm DPM laser (argon 30%) and a 40× objective (HCX PL APO CS 40.0x1.25 OIL UV). The emission filter settings were 500–550 nm for PMT2 (green) and 610–700 nm for PMT3 (red). Depending on the level of zoom used in each image, the XY voxel size was from 100 to 77 nm, approximately. Settings of light and brightness/contrast were adjusted by using the Leica LAS AF Lite imaging software.

When combined with in situ hybridization, immunohistochemistry (IHC) was performed first using RNAse-free or DEPC-treated materials and antibody solutions were supplemented with 100 U/ml RNasin Plus ribonuclease inhibitor (Promega). For IHC, sections were incubated overnight at RT with the appropriate primary antibody solution in PBS supplemented with 0.2% Triton X-100 and 0.1% NaN₃ (PBS-Tx-Az). Primary antibodies used in this study were mouse anti-β3tubulin (1:2000, Promega), rabbit anti-ATF3 (1:200, Santa Cruz Biotechnologies), rabbit anti-CGRP (1:2000, Sigma), mouse anti-NF200 (1:500, Sigma) and rabbit anti-glial fibrillary acidic protein (rabbit anti-GFAP, 1:1000, DakoCytomation). Secondary antibodies were added for 4 h and were donkey anti-mouse AlexaFluor 488 and donkey anti-rabbit AlexaFluor 546 (1:1000, Invitrogen). IB4 detection was performed by using biotin-conjugated IB4 (1:200, Sigma) and AMCA Avidin D (1:400, Vector Labs).