Goat anti rat cy3

The activation of adult‐born cells was examined using immunohistofluorescence. To visualize cells that incorporated thymidine analogs, one‐in‐ten sections were incubated with different anti‐BrdU antibodies (BrdU and CldU, rat primary antibodies at 1/200 Accurate Chemical; IdU, mouse primary antibodies at 1/200, BD Biosciences). Sections were also incubated with Zif268 (rabbit, 1:500, Santa Cruz Biotechnology). Bound antibodies were visualized, respectively, with Cy3‐goat anti‐rat (1:1,000, Jackson) or Cy3‐goat anti‐mouse (1:1,000, Jackson) and Alexa 488 goat anti‐rabbit antibodies (1:1,000, Jackson). CldU‐Zif268 and IdU‐Zif268 labeling were analyzed on different sections because of some cross‐reactivity between secondary antibodies made in mice or rat (Figure 1 in Tronel, Charrier, et al., 2015). All BrdU^‒, CldU^‒, or IdU‐labeled cells expressing Zif268 (one side) were analyzed using a confocal microscope with HeNe and Arg lasers (Leica, DMR TCSSP2AOBS), with a plane apochromatic 63X oil lens (numerical aperture 1.4; Leica). The percentage of BrdU^‒, CldU^‒, or IdU‐labeled cells that expressed Zif268 was calculated as follow: (Nb of Xd ⁺/IEG⁺ cells)/[(Nb of XdU⁺/IEG^‐ cells) + (Nb of XdU⁺/IEG⁺ cells)] × 100. All sections were optically sliced in the Z plane using 1‐µm interval, and cells were rotated in orthogonal planes to verify double labeling.

One‐of‐ten series was incubated with a rat monoclonal anti‐BrdU antibody (1/200, Accurate Chemical) and with a mouse monoclonal anti‐NeuN antibody (1:500, Millipore). Bound anti‐BrdU and anti‐NeuN antibodies were visualized with a Cy3‐goat anti‐rat (1:1,000, Jackson) and an Alexa 488 goat anti‐mouse IgG antibody (1:1,000, Jackson). The phenotype of IdU‐IR cells and CldU‐IR cells was determined using rabbit anti‐calbindin antibodies (1/200, Millipore) that were revealed with Alexa 488 goat anti‐rabbit IgG antibodies (1/500, Jackson). We also analyzed the phenotype of Zif268 cells by incubating one‐in‐ten sections with a rabbit anti‐Zif268 antibody (1:500, Santa Cruz Biotechnology) and a mouse monoclonal anti‐NeuN antibody (1:500, Millipore). Bound anti‐Zif268 and anti‐NeuN antibodies were visualized with a Cy3‐goat anti‐rabbit (1:1,000, Jackson) and an Alexa 488 goat anti‐mouse IgG antibody (1:1,000, Jackson).

Either last-instar larval or 12–24 h pupal wing discs were fixed for 30 min in 0.1 M PIPES (pH 6.9), 1 mM EGTA, 1% Triton X-100, 2 mM MgSO₄, and 1.8% formaldehyde. The discs were then incubated in 50 mM Tris (pH 6.8), 150 mM NaCl, 0.5% NP40, and 5 mg/ml bovine serum albumin (BSA) (block buffer) for a minimum of 2 h at 4 °C. The wings were then placed in 50 mM Tris (pH 6.8), 150 mM NaCl, 0.5% NP40, and 1 mg/ml BSA (wash buffer) containing either rabbit anti-Spalt (1:200), or mouse anti-En/Inv (4F11) (1:5)/rat anti-Ci (1:25)/rabbit anti-Dll (1:100) and incubated overnight at 4 °C. The wings were washed 4 times in wash buffer and then incubated for 2 h at 4 °C in wash buffer containing goat anti-mouse FITC (1:200, Jackson Laboratories, West Grove, PA), goat anti-rat Cy3 (1:200, Jackson Laboratories, West Grove, PA), and goat anti-rabbit Cy5 (1:200, Jackson Laboratories, West Grove, PA). The wing discs were washed four times in wash buffer and then placed on glass slides with the Vectashield (Vector Laboratories, Burlingame, CA). Glass coverslips were applied over the discs and images were collected on a MRC600 laser-scanning confocal microscope. Images were individually collected and then assembled using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA) software.

The following primary antibodies were used for immunostaining of optically cleared tissues: polyclonal rabbit anti‐α‐smooth muscle actin (SMA) (Abcam; ab5694; 1 : 300); and from (BioLegend UK Ltd, London, UK): rat anti‐CD45 clone 30‐F11 (103102; 1 : 300), CD11c clone N418 (117302; 1 : 200) and MHCII I‐A/I‐E clone M5/114.15.2 (107601; 1 : 300). The following macrophage markers were found to be unreliable with the tissue clearing protocols used: rat anti‐F4/80 (clone BM8), rat anti‐CD11b (clone M1/70), rat anti‐CD68 (clone FA‐11) (all from BioLegend) and rat anti‐F4/80 (clone Cl:A3‐1; from AbD Serotec, Kidlington, UK). The following secondary antibodies (all used at 1 : 500) were purchased from Invitrogen: goat anti‐rabbit Alexa Fluor 488 (A11008), goat anti‐rat Alexa Fluor 647 (A21247) and goat anti‐rat Cy3 (A10522); and from (Jackson ImmunoResearch, Ely, UK): goat anti‐Armenian hamster Cy3 (127‐165‐160).
For 2D analysis, SMA expression was detected using a mouse anti‐human SMA primary antibody (clone 1A4, Agilent; M0851) with a peroxidase‐conjugated ImmPRESS anti‐mouse IgG polymer detection kit (Vector Laboratories Ltd; MP‐7402, Peterborough, UK) using standard development with DAB. Mouse IgG1 was used for species‐ and isotype‐matched control (Agilent; X0931).

P1 brains, representing pups from 4–5 independent litters, were fixed in 4% PFA, cryopreserved through incubation in 10%-20%-30% sucrose/1 X PBS gradient over 72 hours at 4°C, mounted in O.C.T. compound and stored at -20°C. Brains were cryo-sectioned coronally at 20 μm over a series of 7 slides, such that each slide contained representative non-consecutive brain sections. Slides were re-hydrated in 1X PBS and incubated in blocking solution containing 2% goat serum/0.01% Triton-X in 1X PBS for 1 hour at room temperature. Antibodies were dissolved in blocking solution and applied over night at 4°C as follows: anti-PECAM1 (rat, 1:50; Thermo Fisher Scientific); anti-PH3 (rabbit, 1:1000; Millipore-Sigma) anti-Satb2 (mouse, 1:250; Abcam); anti-Tbr1 (rabbit, 1:250; Abcam). DAPI (1:4000; Millipore-Sigma) was used to detect cell nuclei. The following secondary antibodies were used: goat anti-rabbit CY3 (1:250; Jackson Immunoresearch); goat anti-mouse Alexa488 (1:1000; Jackson Immunoresearch); goat anti-rat CY3 (1:250; Jackson Immunoresearch).

Pancreas and colon specimens were fixed with 4% paraformaldehyde, incubated with 30% sucrose, embedded in OCT compound (Olympus), and frozen in dry ice. 10-μM sections were permeabilized and blocked with Image-It FX signal enhancer (Invitrogen, Molecular Probes). The following antibodies were used: rabbit anti-GFP (Abcam, 1/100), rat anti-mouse CD8a (BD Pharmingen, 1/100), mouse anti-Cytokeratin 8 (TROMA I, 1/50), goat anti-rabbit Alexa Fluor 488 (Molecular Probes, 1/500), goat anti-mouse Alexa Fluor 488 (Molecular Probes, 1/500), and goat anti-rat Cy3 (Jackson ImmunoResearch, 1/500). Slides were mounted with Vectashield mount medium containing DAPI (Vector Laboratories).