ProLong™ Glass Antifade Mountant with NucBlue™ Stain (Invitrogen™)

ProLong Glass Antifade with NucBlue Stain is a ready-to-use hard-setting mounting media with a refractive index of 1.52 for high resolution microscopy imaging that contains the nuclear counterstain Hoechst 33342 to detect DNA. ProLong Glass Antifade Mountant with NucBlue Stain can be applied directly to fluorescently labeled cells or tissue samples on slides or coverslips. When bound to DNA, NucBlue (Hoechst 33342) is excited by UV light at 360 nm with an emission maximum at 460 nm and is detected using a DAPI traditional filter. ProLong Glass mountant has a refractive index (RI) of 1.52, similar to glass coverslips, compatible immersion oil, and oil-immersion microscope optics, which enables superior resolution and sensitivity. ProLong Glass mounting media is designed to provide unparalleled photobleach protection across the visible and near infra-red spectra. You can use ProLong Glass Antifade Mountant with NucBlue Stain with almost any fluorescent dye or fluorescent protein (e.g., GFP, RFP, mCherry) and with any cell/tissue sample up to 150 microns in thickness. With embedded nuclear DNA counterstain of NucBlue within the ProLong Glass mounting media, multiplexing is smooth and effortless.

ProLong Glass Antifade Mountant with NucBlue Stain is provided in a 2 mL dropper bottle or 10 mL bottle. After removing excess water, just simply apply a drop of mounting media to the sample, add a coverslip, cure, and image. ProLong Glass Mountant with NucBlue Stain minimizes optical refraction and spherical aberration in the optical path, which enables longer-term storage of your samples. ProLong Glass with NucBlue Stain mountant does not discolor or shrink when cured and stored long term and provides nuclear counterstaining directly in the mounting media for DNA detection.

Optimal refractive index for the brightest and sharpest images
The RI determines how much the path of light is bent, or refracted, when entering a material. In microscopy, refractive index differences between specimen and mounting medium should be minimized and can be reduced by using mountants with a higher RI such as ProLong Glass mountant. Depending on your sample type, thickness, imaging depth and/or staining reagents (e.g. fluorescent proteins or dyes) and whether you need a curing or non-curing mountant, choosing the right mounting media will impact image resolution and should be strongly considered for optimal experimental design. For example, when Prolong Glass Antifade Mountant (1.52 RI) is compared with a mounting medium with a refractive index of 1.47, the axial resolution is improved by 32% at a focal depth of 40 μm and 75% at a focal depth of 100 μm. The signal intensity and sensitivity of fluorophores is also improved as the focal depth increases within the sample. Prolong Glass is optimal for acquisition of Z-Stack, 3D, and 2D images of cells and tissue up to 150 μm while providing superior nuclear labeling of most cells and tissues alongside NucBlue stain to assess DNA with nuclear staining.

Prevent photobleaching across the visible and near-IR spectra
Compatible with almost all fluorescent dyes and fluorescent proteins, ProLong Glass Antifade Mountant with NucBlue provides superior protection against photobleaching. ProLong Glass mounting media does not quench fluorophores and provides high-resolution images with a wide array of fluorescent dyes, including Alexa Fluor, FITC, TRTC, Texas Red, Cy3, and Cy5 dyes while NucBlue for nuclear DNA staining is delivered within the mountant. ProLong Glass Antifade Mountant is an ideal reagent for archiving samples and for preserving signal strength during prolonged imaging sessions, such as repeated scanning under confocal laser scanning illumination or with super-resolution microscopy,

Glass mountants are recommended for all immersion oil imaging applications and for samples greater than 10 microns. ProLong Glass Antifade Mountant with NucBlue stain contains an optimized concentration of Hoechst 33342 stain, which provides superior nuclear labeling of DNA within most cells and tissues. However, specimens thicker than 30 μm can require optimization and longer incubations. For specimens thicker than 30 μm, consider pre-incubation with labeling reagents such as NucBlue Live ReadyProbes Reagent followed by mounting with ProLong Glass Antifade Mountant. To image your sample immediately after NucBlue staining, use our SlowFade Glass Antifade Mountant, a non-curing mountant with similar 1.52 refractive index properties.