SlowFade Antifade Reagents

Figure 2. SlowFade Glass mountant retains the best axial/XZ resolution at 150 micron focal depth compared to mountants of lower refractive index. To detect the lateral and axial resolution at shallow and deep focal depths, sub-resolution fluorescent yellow (Ex/Em 505 nm/515 nm) 170-nm microspheres were absorbed onto the surface of a glass coverslip and a microscope slide. Two pieces of tape were stacked and used as spacers to position mounted coverslips (Zeiss™ high tolerance #1.5 170 nm ± 5 nm) approximately 150 µm from the microscope slide. Microspheres were mounted in SlowFade Glass (RI ~1.52) or SlowFade Diamond (RI ~1.42) or VECTASHIELD (R1 ~1.45) and coverslips were adhered to the microscope slides with paraffin. Plotted data shows axial and lateral resolutions as a function of focal depth for microspheres absorbed to the coverslip (0 µm) and microscope slide (150 µm). SlowFade Glass with a refractive index of ~1.52 maintains a higher axial resolution than mountants of 1.42 and 1.45 refractive index at 150 µm focal dept. Lateral resolution remains the same in all mountants at all focal depths tested as expected. The maximum theoretical axial resolution of the microscope is 500 nm, with 200 nm for lateral direction.

Figure 4. Improved focal depth in 100 µm-thick brain tissue sections with SlowFade Glass Antifade Mounting Media. Cryo-preserved 100 µm-thick rat brain sections were stained for GFAP (red) with Rabbit Anti-GFAP (Cat. No. OPA1-06100) and Alexa Fluor Plus 594 Goat Anti-Rabbit (Cat. No. A-32740) overnight. Nuclei (cyan) were stained with DAPI nuclear stain (Cat. No. D1306). Stained samples were mounted with SlowFade Glass (Cat. No. S36917), SlowFade Diamond (Cat. No. S36967), or SlowFade Gold (Cat. No. S36940) non-curing Antifade Mounting Media. Tissue sections were imaged on a Zeiss™ LSM 710 confocal microscope using a Plan-Apochromat 63×/1.4 NA Oil objective sampling at a rate of 71 nm in the x and y dimensions and 100 nm in the z dimension, with a pixel size of 0.07 μm. Z-projections were generated using Zeiss™ Zen software.