Biotinylated goat anti mouse igg secondary antibody

The tumor tissue was fixed in 10% neutral-buffered formalin, embedded in paraffin wax, and cut into sections of 3 μm thickness. Then, the sections were immunohistochemically stained with anti-Ki67 and anti-cleaved (cl)-Caspase-3 antibodies (Cell Signaling Technology) at 4°C overnight. Slides were rinsed with phosphate-buffered saline (PBS) and incubated at room temperature for 30 minutes with biotinylated goat anti-mouse IgG secondary antibody (Dako, Glostrup, Denmark). After washing in Tris-hydrochloride acid buffers (TBS), the slides were incubated with streptavidin-peroxidase reagent (Dako) and treated with 3,3′-diaminobenzidine (DAB; Sigma Aldrich; Merck KGaA) for 5 minutes. Finally, sections were rinsed with ddH2O and counterstained with hematoxylin. Slides were observed under microscope, with the selection of 5 fields of view randomly. The final percentage of positive cells was calculated with the Motic Image software (version 1.2; Micro-Optical Group Co.)

Coronal brain sections, 5 µm thick and mounted onto glass slides (three sections per slide), were deparaffinised, boiled for antigen retrieval in 10mM citric buffer and then incubated in 0.3% (v/v) hydrogen peroxidase solution. Sections were blocked for 20 min in blocking solution (0.1% (w/v) BSA in PBS), incubated in primary antibody (NeuN Clone A60, Merck Millipore, Burlington, MA, USA, diluted 1:11,000), followed by incubation in biotinylated goat anti-mouse IgG secondary antibody (Dako Denmark, Glostrup, Denmark). Primary and secondary antibodies were diluted in blocking solution and reactions were conducted for 1 h at room temperature (RT). Sections were then developed with diaminobenzidine solution (Dako Denmark) and embedded in Neo-Mount (Merck Millipore, USA). Images were taken using a light microscope at a 200× magnification (Carl Zeiss, Jena, Germany) and quantification was conducted by an investigator, blinded with respect to the genotype and the treatment. Neurons expressing NeuN were counted manually in a fixed area in hippocampal CA1, CA3 and dentate gyrus, all at Bregma level −3.16 ± 0.36, as well as in primary motor cortex (MC) at Bregma level 0.74 ± 0.36 [62 ].

PBS-perfused lungs were collected, fixed in 4% paraformaldehyde for 24–48 h, and then stored in 70% ethanol. Dehydrated lungs were embedded in paraffin and cut into 5-µm sections. Lung sections were rehydrated by successive washes in xylene (Sigma-Aldrich), 100% ethanol, 95% ethanol, 70% ethanol, and 50% ethanol. Heat-induced epitope retrieval was performed in citrate buffer (10 mM citrate, pH 6.0), and excess aldehyde was quenched with 0.2 M glycine. After blocking with 10% normal goat serum (Dako), sections were stained with mouse anti-human CD68 antibody (clone PG-M1; Dako), diluted 1:100 in 2% normal goat serum/PBS overnight at 4°C, followed by a biotinylated goat anti-mouse IgG secondary antibody (Dako), diluted 1:200 in 2% normal goat serum/PBS for 1 h at room temperature. Endogenous peroxidase activity was removed by an additional blocking step in 1% hydrogen peroxide/methanol (Sigma-Aldrich). Staining was revealed with the DAB Peroxidase (HRP) Substrate Kit and Vectastain Elite Kit (both from Vector Labs). Finally, slides were counterstained in hematoxylin (Sigma-Aldrich), dehydrated, and mounted using Permount.