Alexa fluor 633

Mice were transcardially perfused with 4% paraformaldehyde (PFA) in phosphate buffer. Brains were postfixed for 48 hours and sectioned on a vibratome (Leica Microsystems) and collected in cold PBS. To count the BrdU in the hippocampal dentate gyrus SGZ, free-floating sections were then incubated with primary antibody for BrdU (1:500; Fitzgerald) and biotin conjugated secondary (Jackson ImmunoResearch, West Grove, PA) along with Alexa Fluor 633 (Invitrogen) was used. Slices were mounted in Vectashield (Vector) and observed with a fluorescence microscope (Olympus Fluoview FV10i, Confocal Laser Scanning Microscope, Center Valley, PA) for labeling at × 10. To determine the number of BrdU+ cells in the SGZ, we counted every sixth section (6 sections per brain) and multiplied the result by 6.^{42 (link)} Representative slices from each group were separately double stained for doublecortin (DCX) and BrdU to determine whether BrdU+ cells were also DCX+. Free-floating sections were incubated with primary antibodies for Brdu (1:500) and DCX (1:300; Millipore). After primary incubation, the corresponding secondary antibody conjugated with Alexa Fluor 633 (Invitrogen) for DCX and biotin conjugated secondary (Jackson ImmunoResearch) along with Alexa 546 (Jackson ImmunoResearch) was used for Brdu.

U251MG cells growing on glass cover slips were synchronized by double thymidine block (2 mM), released for 9 h and treated with DMSO, 100 nM paclitaxel, or 8 μM 6, 11 or 22 for 15 h. Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Digitonin/PBS for 10 min and washed three times with PBS-T (PBS Tween-20). Slides were then blocked in 0.2% bovine serum albumin for 30 min and incubated with anti-β-tubulin and MAP1B antibodies at 1:350 and 1:300 dilution, respectively, in PBS-T for 1 h at room temperature. Subsequently, cells were rinsed three times with PBS-T and incubated for 1 h with Alexa Fluor 546 (donkey anti-mouse) and Alexa Fluor 633 (donkey anti-goat) (Molecular Probes/Invitrogen, Carlsbad, CA) secondary antibodies for β-tubulin and MAP1B detection, respectively, at room temperature in the dark. Specimens were then washed three times with PBS-T, mounted on slides with DAPI Fluoromount-G mounting medium (Southern Biotech, Birmingham, AL), and examined under a Leica TCS SP5 confocal microscope (Leica, Germany). Images were captured and processed using Leica LAS AF Lite software.