Igg isotype control

Formaldehyde-fixed paraffin-embedded sections were steamed in IHC-Tek Epitope Retrieval Solution (IHCWorld) for 40 minutes, then blocked with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% donkey serum. Sections were incubated overnight at 4°C with a goat anti-mouse NPNT polyclonal (R&D, catalog number AF4298) or IgG isotype control (Jackson Immunoresearch, catalog number 005-000-003) at 2 μg/ml in TBST containing 1% BSA. Staining was visualized using Alexa Fluor555-conjugated donkey anti-goat (Invitrogen) antibody at 4 μg/ml and images were captured at an equivalent laser intensity with a Nikon Eclipse Ti-U epifluorescence microscope.

Expression of αvβ3 integrin protein in U251 glioma cells was examined via the immunofluorescence technique. U251 glioma cells on glass coverslips were incubated with medium containing different concentrations of melatonin under normoxia or hypoxia for 24 hours and were then fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. Cells were then incubated with primary antibodies to αvβ3 integrin (1:100, Cell Signaling) overnight at 4°C. Binding specificity was controlled by an IgG isotype control (Jackson Immunoresearch). Subsequently, secondary antibodies Cy3-conjugated AffiniPure donkey anti-rabbit IgG (Jackson Immunoresearch) at 1/500 dilution were applied for 1 hour and viewed using a confocal microscope (Zeiss). αvβ3 integrin staining was presented as the average fluorescence intensity of three pictures per group. All sections were performed in Vectashield Mounting Media with 4′,6-diamidino-2-phenylindole (DAPI).

Formalin-fixed lung tissues were paraffin embedded, and 5-μm-thick sections were prepared, dewaxed in xylene, and rehydrated through graded concentrations of alcohol to water. Then, antigen retrieval was performed in boiling sodium citrate buffer for 10 min. Sections were incubated with blocking buffer for 1h at room temperature, followed by overnight incubation with either anti-mouse Sema3E antibody (R&D Systems, Minneapolis, MN) or isotype control IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa) at 4°C. Slides were then washed twice with TBS followed by incubation for 1h at room temperature with biotin-conjugated secondary antibody. After extensive washing with TBS, slides were incubated with streptavidin-alkaline phosphatase for 30 min at room temperature. Development was performed using Fast Red (Sigma-Aldrich, Oakville, ON, Canada) and counterstained with modified Mayer's hematoxylin (Fisher Scientific, Fair Lawn, NJ). Finally, slides were mounted and visualized using AxioVision software (Carl Zeiss, Inc, Thornwood, NY).