Fluorescein labeling goat anti rabbit antibody

The formalin-fixed paraffin-embedded (FFPE) samples of three subarachnoid and four vesicular cysts were cut into 4 µm sections placed on poly-l-lysine coated slides and treated as previously described^{22 (link),23 (link)}. The slides were submerged in 10 mM citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) for 30 min at 95 °C, incubated for 30 min with a blocking solution (PBS pH 7.2, 0.05% Tween 20, 0.1% Triton X-100, 2% goat serum, 2% BSA) in a humid chamber at RT, then incubated overnight at 4 °C with primary antibody in PBS (Supplementary data 5), washed three times for 2 min with washing solution (PBS pH 7.2, 0.05% Tween 20) and then incubated for 30 min at RT with the fluorescein-labeling goat anti-rabbit antibody (Jackson ImmunoResearch Lab, West Grove, PA, USA) diluted 1/500 in PBS. The sections were then washed with PBS and mounted with VectaShield mounting medium with DAPI (Vector, Laboratories, Burlingame, CA, USA). Images were captured by confocal microscopy (Zeiss, LSM880, Oberkochen, Germany).

The slides were submerged in 10 mM citrate buffer (10 mM citric acid, 0.05% Tween 20, pH 6.0) for 30 min at 95°C, incubated for 30 min with a blocking solution (PBS pH 7.2, 0.05% Tween 20, 0.1% Triton X-100, 2% goat serum, 2% BSA) in a humid chamber at RT, then incubated overnight at 4°C with rabbit anti-phospho-histone H3 (Ser10) antibody (Cell Signaling Technology, Danvers, MA) diluted 1/800 in PBS, washed three times for 2 min with washing solution (PBS pH 7.2, 0.05% Tween 20) and then incubated for 30 min at RT with the fluorescein-labeling goat anti-rabbit antibody (Jackson ImmunoResearch Lab, West Grove, PA) diluted 1/500 in PBS. The sections were then washed with PBS and mounted with VectaShield mounting medium with DAPI (Vector, Laboratories, Burlingame, Ca). The human cell line U251 was included as a proliferation control. Images were captured by confocal microscopy (Zeiss, LSM880, Oberkochen, Germany).

Prior published protocols were employed with some modifications^{22 (link)}. The cells isolated from subarachnoid cysts were fixed with chilled methanol overnight at 4 °C and incubated at 56 °C with a digoxigenin-labeled LNA type probe (argo2; 5ʹTACGACAACGATGAGTTGGAGA-3ʹ) (Qiagen/Life Technologies, Gaithersburg, MD) at a concentration of 0.5 ng/μl designed using the annotated sequence of the gene (TsM_000674100). The samples were incubated overnight at 4 °C with an anti-digoxigenin antibody (1/500) conjugated to peroxidase (Roche) and placed 10 min. in a fluorescein-tyramide developing solution. For phospho-SMAD2 protein detection, cells were incubated overnight at 4 °C with an anti-phospho SMAD2 antibody (1/500). Then for 30 min at RT with the fluorescein-labeling goat anti-rabbit antibody (Jackson ImmunoResearch Lab, West Grove, PA, USA) diluted 1/500 in PBS. Finally, the cells were mounted with VectaShield mounting medium with DAPI (Vector, Laboratories, Burlingame, Ca). Images were captured by confocal microscopy (Zeiss, LSM880, Oberkochen, Germany).