Rat anti ki67

Brains were fixed overnight at 4°C in 4% PFA in PBS, then cryoprotected in 30% sucrose in PBS for 1–3 days at 4°C, then frozen in OCT or cryogel on dry ice after 3–12 hr equilibration at 4°C. Sections were cut at 12 μm thickness spanning the rostral half of the SVZ (typically 6 sections per slide) or the entire olfactory bulb (typically 8 sections per slide). Sections were immunostained with the following primary antibodies: rat anti-BrdU (Abcam clone Bu1/75, 1/500, after heat-mediated antigen retrieval), guinea pig anti-Dcx (1/1000; Millipore), mouse anti-Mcm2 (BD Biosciences, 1/500, after heat-mediated antigen retrieval), rat anti-Ki67 (1/500; eBioscience), mouse anti-tyrosine hydroxylase (1/1000; Millipore), rabbit anti-calretinin (1/1000; Sigma), rabbit anti-calbindin (1/500; Millipore), rabbit anti-S100β (1/1000; Dako), rabbit anti-GST-pi (1/3000; Enzo, Farmingdale, NY), and mouse anti-NeuN (1/1000; Millipore). Fixed whole-mount SVZs were stained with mouse anti-acetylated tubulin (1/1000; Sigma), rabbit anti-β-catenin (1/500; Sigma), mouse anti-GFAP (1/3000; Sigma), and goat anti-EGFR (1/250; R&D Systems). Alexa Fluor 488-, 555-, and 647-conjugated secondary antibodies were used (Life Technologies, Carlsbad, CA).

IHC staining of the tumors was performed as described [27 (link)]. Paraffin-embedded sections were stained ON at 4°C with a rabbit anti-CD31 (4 μg/ml, Abcam) and a rat anti-Ki67 (1 μg/ml, eBioscience) antibody. After washing, sections were incubated for 3 h at RT with Alexa Fluor 568 goat anti-rabbit antibody (4 μg/ml; Molecular probes) and Dylight 650 conjugated goat anti-rat antibody (4 μg/ml; Thermofisher) and examined by fluorescence microscopy.

After culture and imaging, slices were fixed in 4% PFA at 4°C for 4 h, and washed thoroughly in PBS. Slices were blocked in 10% normal horse serum (NHS) and 0.4% Triton-X in PBS for 1 h and incubated in primary antibody (in PBS, 2.5% NHS and 0.4% Triton-X) for 48 h at 4°C. Primary antibodies included: rabbit anti-Olig2 and mouse anti-NeuN (1:500; Millipore, Germany), rat anti-Ki67 (1:500; eBiosciences, San Diego, CA), goat anti-GFAP (1:2000) and goat anti-SOX2 (1:500; Abcam, UK) and rabbit anti-GFAP (1:500; Dako, Denmark). After PBS rinses, slices were incubated in the appropriate secondary antibody conjugated to Alexafluor 405, 594, or 633 (1:500; Life Technologies, Carlsbad, CA) in PBS with 2.5% NHS and 0.4% Triton-X overnight at 4°C. Slices were washed in PBS, placed between two coverslips with a 400 um thick slice of agarose to act as a spacer if needed and mounted in Fluoromount-G (eBiosciences, San Diego, CA).
All fixed tissue was imaged with a laser-scanning confocal microscope (Olympus FV1000, Germany) equipped with a UV diode (405 nm excitation) and three laser lines (488, 543, 633 nm) along with the appropriate emission filter sets; this configuration allowed for imaging of GFP expression and up to three additional markers in a single slice (for example see Figure 4A).

Mice were euthanised, and dissected corneas were fixed in 100% methanol for 1 h at 4 °C and then washed in PBS. Corneal flat mounts were incubated in 20 mM ethylenediaminetetraacetic acid for 60 min at 37 °C and blocked with 3% bovine serum and 0.3% Triton X-100 in PBS for 60 min at room temperature. For immunostaining, tissues were incubated overnight at 4 °C with primary antibody rabbit anti-β tubulin III (1:500; #T2200, Sigma, St Louis, MO, USA) and rat anti-CD45 (1:500; #550539, BD Biosciences, Franklin Lakes, NJ, USA) or rabbit anti-Iba1 (1:500; #019-19741, Wako, Osaka, Japan) and rat anti-Ki67 (1:500; #14-5698-80, eBiosciences, Carlsbad, CA, USA). Afterwards, tissue flat mounts were washed with PBS before incubation with the secondary antibodies, goat anti-rabbit Alexa Fluor 647 (1:500; #A21244, ThermoFisher Scientific, Carlsbad, CA, USA) and goat anti-rat Alexa Cy3 (1:500; #A10522, ThermoFisher Scientific, Carlsbad, CA, USA) and Hoechst (1:1000; Sigma, St Louis, MO, USA) for 120 min at room temperature. Immunostained samples were then washed and mounted onto glass slides with aqueous mounting medium and coverslipped for imaging.

Immunofluorescence was performed as previously described [17 (link)]. Briefly, brains were sectioned at a thickness of 30 µm and left 1 h in blocking solution (10% normal donkey serum, 0.3% Triton-X1000, 0.04% NaN₃ in PBS). Following the blocking step, the sections were incubated with primary antibodies (diluted in blocking solution) overnight at 4 °C. The primary antibodies used were rabbit anti-Ascl1 (Cosmo Bio Co., CAC-SK-T01-003, concentration 1:250), goat anti-Dcx (Santa Cruz, SC-8066, concentration 1:500), mouse anti-NeuN (Millipore, MAB377, concentration 1:500), rabbit anti-S100β (DAKO, Z0311, concentration 1:200, antibody was directly conjugated with fluorophore Alexa-647) and rat anti-Ki67 (eBioscience, 14-5698, concentration 1:1000). Subsequently, the sections were washed three times for 5 min in PBS before incubation with secondary antibodies (1:500 in blocking solution) against the species for the specific primary antibodies and conjugated with the fluorophores Alexa-488, Cy3 and Alexa-647. For Ascl1 detection we used a Tyramide Signal Amplification (TSA) protocol (PerkinElmer, NEL701001KT).