Leica dmrb

All slides were examined with a light microscope Leica DMRB (Bensheim, Germany). Neighboring sections containing RIMLF, INC or vestibular nuclei with y-group were imaged using a slide scanner (Mirax MIDI, Zeiss) equipped with a plan Apo-chromate objective (×20). The digitized images were viewed on a computer with the free software Pannoramic viewer (3D Histech; 1.152.3) at the same zooming magnification. The corresponding detailed views of equally arranged and magnified images were analyzed on the computer screen. Identified CR-positive neurons within the regions of interest were analyzed for the presence of PAV or GAD in the magnified image of the neighboring section using blood vessels as landmarks. Single and double labeled neurons were plotted on the outlines of the respective nuclei of interest with drawing software (Coreldraw 11.0; COREL). The same software was used to label the figures. High power photographs of examples were taken with a digital camera (Pixera Pro 600ES, Klughammer, MarktIndersdorf, Germany) mounted on the microscope and processed with Photoshop 7.0 software (Adobe Systems, Mountain View, CA, USA). The sharpness, contrast, and brightness were adjusted to reflect the appearance of the labeling seen through the microscope.

For studying the expression of pan-cytokeratin (CK) in three GSC PDCs, cells were fixed in 4% paraformaldehyde in PBS for 10 minutes at 37°C. Coverslips were then rinsed three times with PBS, and the cells were permeabilized with PBS containing 0.1% Triton X-100 for 30 minutes at room temperature. After coverslips were rinsed three times with PBS, they were blocked with 5% bovine serum albumin (TBST containing 5% bovine serum albumin) for 15 minutes at room temperature and then incubated with primary antibody (pan-cytokeratin, Sant Cruz, 1:200) for 1 h at room temperature. Then, coverslips were washed three times with TBST and incubated for 30 minutes at room with Alexa-Fluor 488-labelled goat anti-rabbit IgG. After washing three times with TBST, the slides were mounted on VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA), Images were observed by confocal microscopy using a Leica DMRB (Leica, Solms, Germany).

Subcutaneous tumors were collected from euthanized mice and fixed in 4% paraformaldehyde (PFA) solution for overnight at 4°C before embedding into paraffin. Paraffin tissue blocks were then cut with the microtome in 5μm thick sections and then subjected to xylene bath and rehydratation. The sections were washed in phosphate saline buffer (PBS) with 0.25% triton X-100 and boiled in 10 mM sodium citrate buffer (pH6) using microwave for 30 min (OPN, P-H2AX) or water-bath at 95°C for 40 min (Ki67). The sections were blocked for 30 min in PBS-normal serum solution (150μL normal serum and 20μL Tween 20 (Sigma Aldrich) in 10 mL PBS) and incubated with the primary antibodies at 4°C overnight. Following antibodies were used: OPN (Abcam, dilution 1:1000), P-H2AX (ser139, Abcam, dilution 1:3000, 1h at room temperature) and Ki67 (clone MIB-1; Dako, dilution 1:100). Following this, the slides were washed once with the blocking solution and the sections were incubated with biotinylated anti-rabbit/mouse IgG (dilution 1:500) for 30 min. Subsequently, the sections were washed in PBS and incubated in avidin-biotin complex kit for 30 min. Finally, the tissue sections were stained with 3, 3′-diaminobenzidine (DAB) and counter-stained in hematoxylin. Pictures of representative fields were taken under a light microscope Leica DMRB (Leica).