A11077

The antibodies employed for this work were as follows. Primary: Rat anti‐CldU (BioRad, OBT0030G; 1:200), mouse anti‐H2AX‐phosphoSer139 (Millipore, clone JBW301; 1:500) rat anti‐BrdU (Abcam, ab6326; 1:30), mouse anti‐BrdU (Becton Dickinson, 347580; 1:25), mouse anti‐single‐strand DNA (Millipore, MAB3034; 1:25), mouse anti‐UNG (Origene, TA503755; 1:500), rabbit anti‐vinculin (Abcam, ab91459; 1:5,000), rat anti‐tubulin (Abcam, ab6160; 1:5,000). Secondary: Goat anti‐rat Alexa Fluor 488 (Thermo Fisher, A11006; 1:1,000), donkey anti‐mouse Alexa Fluor 568 (Thermo Fisher, A10037; 1:1,000), goat anti‐mouse Alexa Fluor 488 (Thermo Fisher, A32723; 1:25), donkey anti‐rabbit Alexa Fluor 488 (Thermo Fisher, A21206; 1:1,000), goat anti‐rat Alexa Fluor 568 (Thermo Fisher, A11077; 1:25), donkey anti‐mouse Alexa Fluor 647 (Thermo Fisher, A31571; 1:25).

Tissue was dissected in phosphate-buffered saline (PBS), fixed in 4% PFA/PBS for 30′ on ice, washed in PBS-T, incubated with primary antibody in PAXDG (PBS containing 1% BSA, 0.3% Triton X-100, 0.3% deoxycholate and 5% goat serum), followed by washing and incubation with secondary antibody in PAXDG and subsequent mounting in Vectashield with DAPI (Vector Laboratories). Primary antibodies used were: rat anti-myosin (ab51098; Abcam; 1:100) and mouse anti-pERK (M-8159; Sigma; 1:50). Polyclonal rabbit anti-CG1139 (1:200) was purified by New England Peptide Inc., MA, USA. An amino acid sequence 26–40 was selected as the epitope. The following primary antibodies were kindly gifted to us: rat anti-bnl (M. Krasnow; 1:50), guinea pig anti-Path (J. Parrish; 1:200) and rabbit anti-phospho-Drosophila S6 (pS6) (A. Teleman; 1:500). Secondary antibodies used were: Alexa Fluor 488 and 568 conjugated anti-rat antibody (A-11006 and A-11077; Thermo Fisher Scientific; 1:200), Alexa Fluor 568 conjugated anti-mouse antibody (A-11031; Thermo Fisher Scientific; 1:200), Alexa Fluor 568 conjugated anti-rabbit antibody (A-11036; Thermo Fisher Scientific; 1:200) and Alexa-568 conjugated anti-guinea pig antibody (A-11075; Thermo Fisher Scientific, 1:200). Rhodamine phalloidin (R415; Invitrogen; 1:500) was used to visualise F-actin.

Cells were seeded at 1.5 × 10⁵ per 6‐well plate on 13 mm coverslips and the next day treated or not with CldU (1 or 10 μM) or IdU (10 μM). Cells were washed with PBS, fixed in 4% formaldehyde for 15 min, followed by two washes in PBS and denaturation (2 M HCl, 0.5% Triton X‐100) for 30 min. Next, cells were neutralized in 0.1 M Na₂B₄O₇ for 5 min, followed by two washes in PBS. Cells were blocked in BSA in PBS for 1 h followed by incubation with primary rat anti‐BrdU (CldU; Abcam, ab6326, 1:200) or mouse anti‐BrdU (IdU; Becton Dickinson, 347580, 1:200) for 2 h and extensive washing. Samples were incubated in secondary goat anti‐rat Alexa Fluor 568 (Thermo Fisher, A11077; 1:1,000) or goat anti‐mouse Alexa Fluor 488 (Thermo Fisher, A32723; 1:1,000) for 1 h, followed by extensive washing. DNA was counterstained with DAPI and the coverslips were mounted using Mowiol (Sigma Aldrich) mounting media. Images were acquired using an automated Olympus ScanR system, a motorized Olympus IX81 microscope with 40×/0.6 (LUCPLFLN 40× PH) dry objectives and Hamamatsu ORCA‐R2 digital CCD camera C10600. Images were collected and analyzed using ScanR automated software to quantify the fluorescence intensity of individual cell nuclei. All quantification was conducted on cells/samples (typically > 1,000 cells per sample per experiment) selected by scanR software in a random/unbiased manner.

Immunohistochemistry for paraffin embedded-sections using heat-induced epitope retrieval methods were performed as previously described (Magalhães and Rivera, 2014 (link)).
The primary antibodies used in this study were as follows: rat anti-BrdU (1:400, OBT0030G, AbD Serotec), rabbit anti-cleaved caspase-3 (1:50, 9661, Cell Signaling), guinea-pig anti-Dlx2 (distal-less homeobox 2; 1:3000, gift from Dr. Kazuaki Yoshikawa, Osaka University, Osaka, Japan), rabbit anti-Ki67 (1:400, RM-9106-S0, Thermo Fisher Scientific), mouse anti-Nestin (1:400, MAB353, Merck Millipore), rabbit anti-NKCC1 α-wNT (1:200, gift from Dr. Robert James Turner), rabbit anti-Olig2 (1:500, AB9610, Merck Millipore), rabbit anti-phospho-Histone H3 (PH3; 1:1000, 06-570, Merck Millipore), goat anti-Sp8 (1:500, sc-104661, Santa Cruz Biotechnology), mouse anti-TuJ1 (1:5000, MMS-435P, Covance Laboratories).
The secondary antibodies used in this study were as follows: Goat anti-rat Alexa Fluor 568 (1:400, A-11077), Goat anti-guinea-pig Alexa Fluor 568 (1:400, A-11075), Donkey anti-mouse Alexa Fluor 488 (1:400, A-21202), Goat anti-rabbit Alexa Fluor 568 (1:400, A-11011), Donkey anti-goat Alexa Fluor 568 (1:400, A-11057, Thermo Fisher Scientific); Goat anti-rabbit Dylight 488 (1:200, FDR488), Goat anti-rabbit Dylight 549 (1:200, FDR549, Biocare Medical).

Brains were sectioned coronally at 10 μm at approximately bregma −2.00 mm. Serial 10 μm sections immediately rostral and caudal to the section mounted on the Visium Spatial Gene Expression slide (10X Genomics) were collected for immunohistochemistry and stored in cryoprotectant at −20 °C. Primary and secondary antibodies were diluted in 3% normal goat serum (LAMPIRE Biological Laboratories #7332500) with 0.2% Triton X-100 (Sigma CAS #9036-19-5). The tissue was blocked in 10% normal goat serum with 0.2% Triton X-100. Sections were incubated overnight at 4 °C with rabbit anti-P2ry12 (Anaspec #AS-55043A, 1:400), rat anti-GFAP (Invitrogen #13-0300, 1:400), followed by PBS wash and incubation with goat anti-rat AF568 (Invitrogen #A11077, 1:400) and goat anti-rabbit AF488 (Invitrogen #A11304, 1:200) for 2 h at room temperature. The sections were then washed, mounted on slides, and allowed to dry overnight. The slides mounted with dried tissue were then incubated in X-34 (Sigma #SML1954) 10μg/mL solution for 10 min at room temperature before being washed in PBS and differentiated in 80% ethanol for 1 min. The slides were coverslipped with ProLong Gold Antifade Mountant (ThermoFisher #P10144) and imaged on a Zeiss Axio Scan Z1 digital slide scanner at 20× magnification.

Cryosections were stained with lymphocyte antigen 6 complex locus G6D (Ly6G) primary antibody, and a conjugate fluorescent secondary antibody, as previously described [13 (link)]. Cryosections were defrosted at room temperature for 1 h, followed by 10 min at 37 degrees Celsius. The remaining OCT was rinsed with PBS. Tissues were marked with P pap pen, and incubated at room temperature in a wet chamber with blocking solution (5% FCS (biological industries, Beit Haemek)) and 1% Triton ((Sigma-Aldrich, Jerusalem, Israel, CAS 9002-93-1) in pbs) for 1 h. Thereafter, tissues were incubated overnight at 4 °C in a wet chamber with the primary antibody vs. Ly6G (BD biosciences, 551459) diluted at a ratio of 1:100 in blocking solution. PBS rinses were performed, and the tissues were incubated for 2 h at room temperature in a wet chamber with the secondary antibody (Invitrogen A11077) diluted at a ratio of 1:200 in blocking solution. Slides were rinsed, stained with 5 µM DAPI (abcam AB-ab228549) at room temperature for 10 min, rinsed, and covered with a coverslip with mounting medium (VECTASHIELD^® Mounting Medium (H-1000, Zotal)). Images were obtained with NIKON-Ti X40 magnification (N.A. 0.75 dry), using Photometrics Prime 95B camera.