Ab16645

Mice were deeply anesthetized and transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in PBS. For fixation, the brains were kept overnight in 4% paraformaldehyde. Each brain sample was placed in 30% sucrose in PBS for 48 h. After embedding and freezing, each brain was cut into 30 µm coronal slices using a cryostat (CM1950, Leica, Heidelberger, Germany).
For immunostaining, the brain slices were washed three times with PBS and incubated with primary antibodies (Foxp1: rabbit anti-Foxp1, 1:20,000, ab16645, Abcam, USA; GABA: rabbit anti-GABA, 1:1000; PA5-32241, Invitrogen, USA; Orexin: mouse anti-orexin-A,1:600, sc-80263, Santa Cruz Biotechnology, USA; MCH: rabbit anti-melanin-concentrating-hormone, 1:1000, M8440, Sigma-Aldrich, USA; Glutamate: rabbit anti-glutamate, 1:1000, G6642, Sigma, USA; and Biocytin: 1:1000, S21374, Invitrogen, USA) dissolved in PBST (0.3% Triton X-100 in PBS) overnight at 4 °C. The next day, slices were washed with PBS and incubated with secondary antibodies (Donkey anti-rabbit/goat, 1:1000; Jackson ImmunoResearch, USA) for 2 h. Fluorescence images were captured using a confocal microscope (Nikon AIR-MP).

The Immunohistochemistry (IHC) experiments were performed as our previous reported^{10 (link)}. Briefly, the paraffin-embedded tumor tissues were cut into 4 μm. The tissues were retrieved in citric acid buffer (PH = 7.0), and then were blocked with normal goat serum (Boster Biological Technology, Wuhan, China) for 60 min at room temperature. The tissues were incubated with primary antibody (anti-Foxp1, cat no. Ab16645, 1:200 dilution, Abcam; anti-Ki67, cat no. Ab92742, 1:500 dilution, Abcam) overnight at 4 °C. The tissues were then then with a horseradish peroxidase (HRP) -conjugated anti-rabbit secondary antibody for 2 h at 37 °C. Finally, the sections were counterstained with hematoxylin.
The expression of Foxp1 and Ki67 was semi-quantitated by immunoreactivity scoring. The intensity of Foxp1 staining was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (intense) by two pathologists who were blinded to the experiments. The immunoreactivity score was calculated as the percentage of positive cells multiplied by the intensity of staining.

The following primary antibodies and dilutions were used for immunocytochemistry: Wnt3 (1:250, rabbit; LifeSpan BioSciences #LS-C774904, Seattle, WA, USA), Fzd7 (1:50, goat, Thermo Fisher #PA5-47232, Waltham, MA, USA), Foxp1 (1:1000, rabbit; Abcam #ab16645, Cambridge, MA, USA), Foxp1 (1:250, mouse; lab stock), Foxp2 (1:250, rabbit; Abnova #PAB12684, Taipei, Taiwan), Foxp2 (1:250, goat; Santa Cruz Biotechnology #sc21069, Dallas, TX, USA), Gapdh (1:300, mouse, Millipore Sigma #MAB374, St. Louis, MO, USA), GFP (1:1000, chicken; Aves Labs #GFP-1020, Tigard, OR, USA), RFP (1:1000, rabbit; Antibodies-Online #ABIN129578, Limerick, ME, USA). Appropriate species-specific Donkey secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were used at a 1:250 dilution.

Brain sections were prepared as described for in situ hybridization. The sections were washed with TBST (10 mM Tris pH7.4, 150 mM NaCl, 0.1% Tween20), permeablized with 100% methanol at -30°C for 15 min, and then reacted with the primary antibodies at 4°C overnight. The following antibodies were used: mouse anti-Cadherin7 antibody [CCD7-1; Developmental Studies Hybridoma Bank (DSHB), Iowa, IA, USA], rat anti-Ctip2 antibody (ab18465; Abcam, Cambridge, UK), rabbit anti-Foxp1 antibody (ab16645; Abcam), mouse anti-β (III)-tubulin antibody, TUJ1 (MMS-435P; Covance, Princeton, NJ, USA), and rabbit anti-phospho-Histone H3 antibody (06-570; Millipore, Billerica, MA, USA), After washing with TBST, the sections were stained with anti-mouse IgG antibody conjugated with Alexa Fluor 488 (A11029; Life Technologies), anti-rat IgG antibody conjugated with Alexa Fluor 488 (A11006; Life Technologies) or anti-rabbit IgG antibody conjugated with Cy3 (711-165-152; Jackson ImmunoResearch, West Grove, PA, USA). They were then washed with TBST again, and coverslipped with 90% glycerol in PBS.

To validate the localization of the injected virus construct, triple immunohistochemistry analyses were conducted on cryostat sections. Slices were fixed in 4% PFA in 1× PBS for 10 min at 4°C and blocked in 10% ROTI Histol (Carl Roth) solution between stainings. The following antibodies were used: a mouse monoclonal (JC12) antibody against FoxP1 (1:100, ab16645, Abcam, Cambridge), a goat antibody against zRalDH (1:50, sc.22 591, Santa Cruz, Dallas) to delineate HVC, and a rabbit GFP antibody (1:100, ab6556, Abcam, Cambridge) to increase signal strength from virally transmitted GFP. Ultimately, slices were counterstained with Hoechst (Sanofi) and mounted for fluorescence imaging (Apotome, Zeiss).

Immunohistochemistry and 5-bromo-4-chloro-3-indoyl-D-galactopyranoside (X-gal) staining were performed as previously described [20 (link), 41 ]. Tissues were sliced into to 12 or 20 μm thick sections. The following primary antibodies were used to perform immunofluorescence labeling: mouse anti-CRE (Millipore, 69050-3), rabbit anti-EBF1 (Merck, AB10523), rabbit anti-ISL1 (Abcam, ab20670), chicken anti-GFP (AVES Labs, GFP-1020), rat anti-BCL11B (Abcam, ab18465), rabbit anti-FOXP1 (Abcam, ab16645) and rabbit anti-SP9 (1:500) [20 (link)]. Alexa Fluor 488-, Cy3- or 647-conjugated secondary antibodies were purchased from Jackson ImmunoResearch.

For immunohistochemistry the following primary antibodies were used at the indicated dilutions: Isl1 (40.2D6, 1:100, DSHB), Isl1/2 (39.4D5, 1:200, DSHB), Nkx2.2 (74.5A5, 1:100, DSHB), Olig2 (AB9610, 1:100, Millipore), Pax7 (1:10, DSHB), anti-h/m/r Hif-1a (AF1935, 1:100, R&D Systems), pAb anti-Carbonic Anhydrase IX/CA9 (NB100-417, 1:100, Novus Biologicals), TER-119 (MAB1125, 1:100, R&D Systems), rabbit anti-FoxP1 (ab16645, 1:1.000, Abcam), mouse anti-neurofilament-M (RMO 270, 1:1.500, ThermoFischer), anti-mouse Flt1 (103-M31, 1:100, ReliaTech GmbH), En-1 (4G11, 1:50, DSHB). The secondary antibodies that were used were donkey anti-mouse Alexa488 (715-545-150, 1:400, Jackson ImmunoResearch), goat anti-mouse Alexa488 (115-545-146, 1:400, Jackson ImmunoResearch), goat anti-rat Alexa568 (A11077, 1:400, Invitrogen), donkey anti-rabbit Alexa647 (711-605-152, 1:400, Jackson ImmunoResearch). Blood vessels were visualized using Isolectin GS-IB₄ Alexa Fluor 568 conjugate (I21412, 1:250, Invitrogen). Images were collected on a confocal microscope (Zeiss LSM 510 unit mounted on an Axiovert 200 M inverted microscope) with 10x/0,3 EC Plan-NEOFLUAR Objective and/or with 20x/0,8 Plan-APOCHROMAT and on a Nikon AR1 confocal microscope with 40x/1,3 Plan-Fluor Objective. Image processing was performed using Zen 2011 and the NIH ImageJ software.