Anti olig2

To combine the padlock detection and immunohistochemistry, we first hybridized slides with padlock probes as described above. After that, the slides were washed three times for 10 min in PBS and blocked for 1 h in 0.25 % bovine serum albumin in PBS with 0.25 % Triton X-100. Each of the following primary antibodies: anti-NeuN (mouse, 1:1000; Abcam), anti-Olig2 (goat, 1:20; R&D Systems), anti-Sox10 (goat, 1:20; R&D Systems) and anti-Islet 1 (goat, 1:20; R&D Systems) was incubated overnight at 4 °C. After washing with PBS, the corresponding secondary antibodies were applied for 4 h at room temperature: Alexa 594 goat anti-rabbit, FITC goat anti-mouse, Alexa 488 donkey anti-goat and Alexa 597 goat anti-chicken (1:400; Life Technologies). Immunolabelled cells were mounted with Dako ultramount aqueous permanent mounting medium (Dako). Cell imaging was performed using epi-fluorescence microscope Ziess (Zeiss Axio Imager.Z2 epi-fluorescence microscope, filter cubes in Additional file 10: Table S6). The images were assembled in Photoshop CS6 (Adobe).

Forty-μm thick free-floating sections were washed in 0.01 M tris-buffered solution (TBS; pH 7.4) 3 X for 5 min. The tissue then underwent a sodium citrate antigen retrieval (0.1M; pH 7.3) process for 30 min at 37 °C. After antigen retrieval the free-floating sections were washed and blocked in 5% normal serum for 1–2 h. The sections were thereafter incubated in 1% serum in TBS-Triton (TBST) primary antibody solution consisting of one of the following antibodies: anti-Iba1 (1:500, WACO), anti-Olig2 (1:250, clone SP07-02; R&D), anti-GFAP (1:500, clone DIF48; Abcam), anti-CD3 (1:500, clone 17A2; Thermo Fisher), anti-CD8 (1:500, clone 4SM15; eBioscience), anti-Thy1.1 (1:500, OX-7; Invitrogen) anti-CD4 (1:500, clone RM4-5; Thermo Fisher), anti-NK1.1 (1:250, clone EPR22990-12; Abcam). After an overnight incubation, the free-floating sections were washed and put into a 1% serum TBST secondary solution for 2 h. Sections were mounted onto coated glass slides, and cover slipped using hard set mounting medium (Vector Laboratories). Fluorescence images were collected on a Ti2 Nikon microscope using a Ci2 confocal system.

Morphologic examination was performed on Hematoxylin- and Eosin-stained sections (3–4 μm). Immunolabeling was performed using an automated system (Autostainer Dako, Glostrup Denmark) with the following primary antibodies: anti-Ki67 (MIB-1, Dako, prediluted), anti-Olig2 (goat polyclonal, R&D Systems, 1:500) and anti-GFAP (Dako, 1:4000). Deparaffinization, rehydration and antigen retrieval were performed using the pretreatment module PTlink (Dako). SSADH immunostaining was achieved following overnight incubation with anti-SSADH (Novus Biological, 1:100) at 4 °C. Immunostaining was scored by a pathologist (FBV).