Dako target retrieval solution ph 6

Mice xenografts were immediately fixed with 4% formaldehyde and embedded in paraffin. IHC for BAX and BCL-w was performed on 5-μm-thick paraffin-embedded sections from tumor biopsies from mice treated with exosomes or with PBS (control). Isotype-matched irrelevant antibodies were used as a negative control. Following rehydration, the antigen was unmasked for 45 minutes in a 95°C microwave with Dako Target Retrieval Solution (pH 6; Dako, Carpinteria, California, USA). Endogenous peroxidase was blocked for 10 minutes with Dako peroxidase blocking reagent, and non-specific binding was blocked for 20 minutes with Dako protein block. The primary antibodies anti-human BAX and BCL-w (1:100 diluition, Santa Cruz Biotechnology) were added and incubated for 1 hour at room temperature. For control staining, primary antibodies were replaced with irrelevant isotype-matched antibodies (AbCam, Cambridge, UK). The slides were then incubated for 30 minutes with peroxidise-conjugated Dako EnVision polymer, and peroxidase activity was visualized with diaminobenzidine chromogen (Dako). Slides were lightly counterstained with hematoxylin before dehydration and mounting in DePex (VWR International, Oslo, Norway).

Tissue immunofluorescence was realized as described^{20 (link)}. Briefly, 4 µm paraffin-embedded tissue section placed on a glass slide was de-paraffinized in three baths of xylene for 5 min and rehydrated in a series of ethanol baths. For antigen retrieval, slides were incubated in DAKO Target Retrieval Solution, pH6 (DAKO, Carpinteria, California, USA), for 20 min in a microwave oven. Slides were incubated with mouse anti-CFP (Santa Cruz Biotechnology, Dallas, Texas, USA, sc9996, dilution 1/50) and rabbit anti-RFP (Abcam, Cambridge, UK, ab62341, dilution 1/50) primary antibodies for 1 h at RT after proteolytic epitope retrieval in citrate buffer (pH 6.0). Then, Alexa Fluor 488 goat anti-mouse (Molecular Probes, Eugene, Oregon, USA, A11001, dilution 1/400) and 647 goat anti-rabbit (Molecular Probes, A21245, dilution 1:400) secondary antibodies were incubated for 1 h at RT. Slides were mounted using the Vectashield mounting medium plus DAPI (Vector Laboratories). Images were acquired on a Zeiss Cell Observer microscope (Zeiss, Oberkochen, Germany).