RH 795

RH 795 (7-25 µg/mL) potently labels the cell membranes of inner hair cells, outer hair cells, and associated nerve fibers in unfixed ex vivo cochlear specimens from Balb/c mice, enabling high-resolution visualization via confocal microscopy^[2].

Guide (The following is our recommended protocol. This protocol serves only as a guideline and should be modified according to your specific needs).
Ex vivo Live Mouse Temporal Bone^[2]
1. Reagent Preparation
1.1 Stock solution: Weigh RH 795 powder and dissolve it in anhydrous DMSO with gentle vortexing to prepare a 1-2 mg/mL (approximately 2-4 mM) stock solution.
1.2 Working solution: Dilute the stock solution with tissue culture medium to a final concentration of approximately 7-25 μg/mL.
Co-staining with Calcein AM (HY-D0041) (for double labeling if needed): Use a final concentration of 20-60 µg/mL for Calcein AM.
2. Procedure
2.1 Preparation: Dissect out the left temporal bone of a mouse, fix it on a plastic holder, open the bony structure of the apical turn to expose the organ of Corti, and insert a perfusion tube into the scala tympani.
2.2 Perfusion system: Connect to a medium-containing reservoir bottle, and perform continuous perfusion of the scala tympani via hydrostatic pressure difference, allowing the medium to flow out through the opening of the apical turn.
2.3 Pre-staining with Calcein (optional for co-labeling): Perfuse with medium containing Calcein (20-30 µg/mL) for several minutes to ~15-30 min to label the cytoplasm of live cells green.
2.4 RH 795 staining: Switch the perfusion solution to fresh medium containing RH 795 (7-25 µg/mL) and perfuse for 15-30 min
Alternatively, add both dyes to the medium simultaneously for double-labeling perfusion
2.5 Washing/Equilibration: Briefly (for several minutes) switch to dye-free medium or continue perfusion to maintain oxygenation, followed immediately by confocal observation
2.6 Imaging parameters
RH 795: Excitation at 568 nm/ long-pass filter ≥ 560 nm (red channel)
Calcein: Excitation at 488 nm/ band-pass filter at 515 nm (green channel)
Reduce laser intensity appropriately to avoid phototoxicity (the membrane-bound dye RH 795 causes relatively strong photodamage).

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.