Custom probes designed for hybridization with porcine ISG15 were purchased (Affymetrix). Probes were hybridized to formalin-fixed and paraffin-embedded, 5-μm sections of porcine ileum mucosa (ViewRNA in situ hybridization kit, catalog QVC0001; Affymetrix) using the manufacturer protocol for 1-plex hybridization. Slides were deparaffinized using xylene (30 minutes) and heat and protease K pretreated (10 and 30 minutes, respectively). After initial hybridization, signal amplification was performed using pre-amplifier and amplifier probes using a slide hybridization and denaturation system (ThermoBrite, catalog 23-021-580; Leica Microsystems, Wetzlar, Germany). To detect the ISG15 target, slides were incubated with fast-red chromagen (1:30 dilution for 45 minutes), washed, and coverslipped using mounting media containing DAPI (VECTASHIED antifade mounting medium with DAPI, catalog H-1200-10; Vector Labs, Burlingame, CA). Slides were imaged using a fluorescence microscope (Leica DB 5000B with LASX software; Leica Microsystems). Rat ubiquitin probes were hybridized to formalin-fixed and paraffin-embedded rat kidney slides as a hybridization control.
Vectashied antifade mounting medium with dapi
Manufactured by Vector Laboratories
VECTASHIELD antifade mounting medium with DAPI is a ready-to-use solution designed to protect fluorescent signals from photobleaching. It contains the DNA-binding dye DAPI, which selectively labels nuclei.
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2 protocols using vectashied antifade mounting medium with dapi
1
In Situ Hybridization of Porcine ISG15
2
Immunofluorescence Analysis of DNA Damage
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Cells were seeded in 8-well chamber slides, irradiated with 6 Gy and were fixed with 4% formaldehyde. After blocking with 5% normal goat serum (Cell Signaling Technology), slides were incubated with pH2A.X primary antibody (RRID : AB_2118010) or IgG isotype control antibody (RRID : AB_1550038) overnight at 4°C. Slides were incubated with secondary antibody (goat anti-rabbit IgG AF488, RRID : AB_1904025) for 1 h at room temperature. All antibodies were purchased from Cell Signaling Technology. Slides were covered with Vectashied antifade mounting medium with DAPI (Vector Laboratories). Detection was performed on an inverse fluorescence microscope (AxioImager Z1; Zeiss).
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