Goat anti rat 488

Immunofluorescent staining (IF) for CD31 was performed on 10–20 μm cryo-frozen tissue sections. Tissue sections were fixed in 4% paraformaldehyde for 30min, washed, blocked, and incubated with primary antibody (rat anti mouse CD31, 1:50 dilution; BD) overnight at 4°C, followed by successive PBS washes and application of secondary antibodies (1:500, goat anti-rat 488, Invitrogen), followed by PBS washing and mounting with mounting media containing Propidium Iodine or DAPI (4’, 6-diamidino-2-phenylindole; Vectashield, Vector Laboratories) and analyzed using a Nikon confocal microscope Eclipse 80i. Staining for Ki67 was performed on 5-micron sections of formalin fixed, paraffin-embedded mouse ears at pre-determined time points. Sections were de-paraffinized, antigens unmasked in citrate buffer (pH = 6.0), blocked in normal goat serum, and incubated overnight at 4°C in primary antibody (rabbit polyclonal Ki-67, 1:150 dilution, AbCam). Sections were then stained in secondary rabbit anti-goat (Vector Labs), followed by PBS washing and color retrieval using DAB chromagen (Dako). Sections were dehydrated, counterstained using hematoxylin, rinsed in xylene, and coverslipped with cytoseal. H&E staining was performed over 5-μm paraffin section using a progressive method.

After retrograde labeling, the fixed brains were processed using a standard immunocytochemistry protocol: Fixed brains were blocked for 20 minutes in a solution of 5% normal goat serum (NGS) and 0.4% PBST. The brains were incubated with primary antibodies in 5% NGS blocking solution at 4 degrees for 1–3 days. Brains were next rinsed 3 × 10 minutes in PBST and placed in secondary antibodies in 5% NGS blocking solution at 4 degrees for 1–3 days. Finally, brains were rinsed 3 × 10 minutes in PBST and mounted in Vectashield (Vector Labs) for confocal imaging. Primary antibodies used were mouse anti-nc82 (1:40, DSHB), rat anti-mCD8 (1:40, Invitrogen MCD07800), rabbit anti-GFP (1:500, Invitrogen A11122), and rabbit anti-GABA (1:100, #A2052; Sigma, St. Louis, MO). Secondary antibodies used were goat anti-mouse 633 (1:400, Invitrogen A21050), goat anti-rat 488 (1:400, Invitrogen A11006), goat anti-rabbit 488 (1:500 when amplifying anti-GFP, 1:250 when amplifying anti-GABA; Invitrogen A11034) and streptavidin 568 (1:500, Invitrogen S11226).

Brian tissue sections were incubated overnight with primary antibodies at 4°C and then with corresponding fluorochrome-conjugated secondary antibodies at room temperature for 1 h. The following primary antibodies were used: anti-human NKp46 (195314; R&D Systems), CD49a (SR84; BD Bioscience), CD57 (MA1-81071; Invitrogen), CD68 (ED1; Abcam), CD4 (H-370; Santa Cruz), CD8 (UCH-T4; Santa Cruz), CD19 (HIB19; BioLegend), CD16b (CLB-gran11.5; BD Bioscience), CD66b (polyclonal; Bioss), perforin (dG9; BioLegend), CD69 (D-3; Santa Cruz), Caspase-3 (9661; CST), CD31 (JC/70A; Abcam); anti-mouse NKp46 (M20; Santa Cruz), CD49a (Ha31/8; BD Bioscience), ly6G (1A8; BioLegend), TMEM119 (28-3; Abcam), CD68 (ED1; Abcam), CD8 (53-6.7; eBioscience), CD4 (GK1.5; eBioscience), CD19 (1D3; BD Bioscience), CD31 (polyclonal; Abcam), claudin5 (4C3C2; Invitrogen), and ZO-1 (ZO-1-1A12; Invitrogen). The following fluorochrome-conjugated secondary antibodies were used: donkey anti-rabbit 488 (1:1,000; Invitrogen), donkey anti-rabbit 546 (1:1,000; Invitrogen), donkey anti-goat 546 (1:1,000; Invitrogen), donkey anti-mouse 594 (1:1,000; Invitrogen), donkey anti-mouse 488 (1:1,000; Invitrogen), goat anti-rat 488 (1:1,000; Invitrogen), and goat anti-rat 555 (1:1,000; Invitrogen). Images were acquired on a fluorescence microscope (Olympus BX-61).

When necessary, depending on the tissue penetrance and antigen recognition ability of the antibody used, antigen retrieval was performed by microwaving slides in 10 mM sodium citrate buffer for 1 min at maximum power, followed by 10 min at minimum power. Slides were then washed in 1× PBS and incubated in blocking solution (5% normal donkey or normal goat serum, 0.2% Triton^® X-100 in PBS) for 1 h at room temperature. This was followed by incubation in primary antibody overnight at room temperature. Slides were washed in 1× PBS and incubated with secondary antibody solution for 1 h at room temperature. Slides were mounted in Vectashield with DAPI (Vector Laboratories). Primary antibodies used were as follows: rabbit anti-oligodendrocyte transcription factor 2 (1:300, AB9610, Millipore, Burlington, MA), rabbit anti-phosphorylated histone 3 (1:500, 06-570, Millipore), rat anti-somatostatin (1:50, MAB354, Millipore), rabbit anti-parvalbumin (1:1000, PV25, Swant, Marly, Switzerland), rabbit anti-calretinin (1:1000, Swant, 769913), and rabbit anti-Tbr1 (1:1000, gift from the Hevner laboratory, University of Washington School of Medicine, Seattle, WA). The following secondary antibodies were used: (1:250 dilution, Thermo Fisher Scientific): donkey anti-rabbit 555 (A31572), goat anti-rabbit 546 (A11035), goat anti-rabbit 488 (A11008) and goat anti-rat 488 (A11006).

Tissues and organoids were fixed with 4% paraformaldehyde (Electron Microscopy Science) overnight at 4 °C. Tissues were embedded in OCT compound (Fisher Scientific) and stored at − 80 °C. Frozen blocks were cut at 10 µm thickness, and sections were collected onto Superfrost Plus slides (VWR international). Tissue sections and organoids were permeabilized with 0.5% triton X-100 (Sigma Aldrich) in PBS 1X at RT for 40 min and incubated with a blocking buffer containing 3% bovine serum albumin (BSA, Sigma Aldrich) in PBS 1× RT for 40 min. Samples were incubated overnight at 4 °C with primary antibodies diluted in 0.1% triton X-100, 1% BSA in PBS 1×, washed, incubated with secondary antibodies for 1 h at room temperature, washed, and stained for 20 min with 1 µg/mL DAPI (Invitrogen). Finally, slides were mounted with ProLong™ gold antifade mounting medium (ThermoFisher). The following antibodies were used: anti-CD34 coupled with eF660 (clone RAM34) (eBioscience), anti-E-cadherin (clone ECCD-2) (Takara), goat anti-rat488 (Thermo Fisher), anti-Pdpn (gift from A. Farr, University of Washington, Seattle), and goat-anti-hamster 546 (Thermo Fisher), anti-Ki67 coupled with eF660 (clone SolA15) (eBioscience), phalloidin coupled with AF488 (Thermo Fisher).

Bromodeoxyuridine (BrdU) was purchased from Sigma-Aldrich (St. Louis, MO, USA). AG490 was purchased from Calbiochem (San Diego, CA, USA). The following primary antibodies were used in these experiments: rat monoclonal anti-BrdU (Abcam, ab6326, 1:500), mouse anti-Sox2 (Santa Cruz, sc365823, 1:200), rabbit anti-Ki67 (Millipore, AB9260, 1:200), mouse anti-nestin (Abcam, ab6142, 1:200), rabbit anti-doublecortin (DCX) (Abcam, ab18723, 1:400), rabbit anti-glial fibrillary acidic protein (GFAP) (DAKO, 1:1500), rabbit anti-NeuN (Abcam, ab177487, 1:500), rabbit anti-Phospho-Stat3 (Tyr705) (Cell Signaling Technology #9145, 1:2000), and mouse anti-GAPDH (Abcam, ab8245, 1:10000). All secondary antibodies for immunofluorescent study were Alexa Fluor antibodies from Thermo Fisher Scientific (Waltham, MA, USA), namely goat anti-rat 488, goat anti-mouse 568, and goat anti-rabbit TRITC (T2769). All secondary antibodies for Western-blot were ECL™ anti-rabbit and anti-mouse IgG from GE Healthcare (UK) Company.