Biotinylated goat anti rabbit antibody

Glioblastoma cells were stained as published before [104 (link)]. Ki67 was used for the assessment of proliferation, and cleaved caspase 3 for apoptosis detection (Table 1). The labeling of incorporated BrdU was visualized with an anti-BrdU antibody (1:100, DAKO, Aligent, Santa Clara, CA, USA) (Table 1), which was applied for 1 h. For all antibodies and all immunohistochemical stainings, the subsequent steps were identical. After washing with PBS, a biotinylated goat anti-rabbit antibody (1:100, Sigma Aldrich) was applied for one hour, the cells were washed three times with PBS and incubated with Streptavidin for one hour (1:100, Sigma Aldrich). After washing with PBS and Tris buffer, the slides were stained with DAB (Sigma Aldrich) and Hematoxylin (Merck Millipore) and covered with Entallan (Merck Millipore). Propidium iodide-labeled cells were additionally stained with Sytox Green (1:10 000, S7020, Thermo Fisher) for 5 min before covering with DAKO mounting medium (DAKO).
For assessing cell death and proliferation at least 100 cells were counted and 5 areas for each cover slip were recorded with an Axioplan (Zeiss, Oberkochen, Germany) and analyzed using ImageJ v1.46r (National Institutes of Health, Laboratory for Optical and Computational Instrumentation, University of Wisconsin, Madison, WI, USA).

In order to inhibit endogenous peroxidase activity, sections were incubated in methanol containing 3% hydrogen peroxide for 30 min. After washing in phosphate buffered saline (PBS), pH 7.4, sections were incubated in 10% normal goat serum for 1h. Then, sections were incubated overnight with a previously characterized GFAP polyclonal primary antibody (Dako, USA, dilution 1:500). The following day, sections were incubated in biotinylated goat anti rabbit antibody (Sigma Chemical Co.,MO., USA; dilution 1:500). Following this, sections were incubated in ExtrAvidin-Peroxidase® complex (Sigma Chemical Co., MO., USA; dilution 1:500). All antisera were diluted in phosphate-buffered saline (PBS) containing 0.2% Triton X-100 and, in all but in the peroxidase complex, 1% normal goat serum. Incubations in primary antibody were performed overnight at 4°C while incubations in biotinylated antibody, ExtrAvidin-Peroxidase® complex were performed at room temperature (RT) for 1h. Controls were performed by omitting primary antibodies. Development was performed using the DAB/nickel intensification procedure [43 (link)].

Sections were incubated overnight with an Iba1 rabbit polyclonal antibody (Thermo Fisher Scientific Inc., USA, dilution 1:125). The following day, sections were incubated in biotinylated goat anti rabbit antibody (Sigma Chemical Co.,MO., USA; dilution 1:125), and later in Streptavidin-Alexa Fluor® 635 conjugate (Thermo Fisher Scientific Inc., USA, dilution 1:50). Incubations in biotynilated goat anti rabbit antibody and Streptavidin-Alexa Fluor® 635 conjugate were performed at RT for 1 h. Finally, sections were counterstained with Hoechst 33258 (Sigma Chemical Co., MO., USA) and were observed using an Olympus IX-81 inverted microscope.

Endogenous peroxidase activity was inhibited by incubation in methanol containing 3% hydrogen peroxide for 30 min. Overnight incubation with GFAP polyclonal primary antibody (Dako Ink, Cat #Z0334, United States, dilution 1:500) was performed at 4°C. Then sections were incubated with biotinylated goat anti-rabbit antibody (Sigma Chemical Co.,MO; Cat #B8895, dilution 1:500) at room temperature (RT) for 1 h followed by ExtrAvidin-Peroxidase^® complex (Sigma Chemical Co., MO., United States; Cat E2886, dilution 1:500) at room temperature (RT) for 1 h as well. Development was performed using the DAB/nickel intensification procedure (Hancock, 1984 (link)). Controls were performed by omitting primary antibodies.