Jan 18, 2026
Version 2
Staining Murine Whole Lenses with SynaptoRed™ C2 V.2
- Rylee E. King1,
- Justin Parreno1,2
- 1Department of Biological Sciences, University of Delaware, Newark, DE, USA.;
- 2Department of Biomedical Engineering, University of Delaware, Newark, DE, USA.
Protocol Citation: Rylee E. King, Justin Parreno 2026. Staining Murine Whole Lenses with SynaptoRed™ C2. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk14r5g5r/v2Version created by Rylee E King
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 16, 2026
Last Modified: January 18, 2026
Protocol Integer ID: 238802
Keywords: spatial organization in whole lens tissue, murine whole lenses with synaptored, staining murine whole lens, whole murine lens, whole lens tissue, intact murine lens, confocal microscopy, nuclear organization within the intact lens, assessing cell morphology, resolution confocal microscopy, intact lens, clear visualization of cellular boundary, cell morphology, cellular morphology, studying tissue architecture, protocol for fluorescent staining, fluorescent staining, tissue architecture, cytoskeletal organization, using synaptored tm c2, cellular boundary, synaptored tm c2, plasma membrane, staining approach
Abstract
Visualization of cellular morphology in intact lenses is essential for studying tissue architecture and cytoskeletal organization. Here, we describe a protocol for fluorescent staining of whole murine lenses using SynaptoRed TM C2 and Hoechst to label plasma membranes and nuclei, respectively. Following ex vivo incubation in Medium 199, intact murine lenses were briefly stained with SynaptoRed TM C2 and Hoechst, washed, and imaged immediately using high-resolution confocal microscopy. This staining approach allows clear visualization of cellular boundaries and nuclear organization within the intact lens without the need for fixation or sectioning. Overall, this protocol provides a rapid and reliable method for assessing cell morphology and spatial organization in whole lens tissue.
Materials
SynaptoRed™ C2, Hoechst (Biotium), PBS, medium 199
Troubleshooting
Stain Preparation
SynaptoRed™ C2 stock was made to 10 mM, intermediate stock of 0.1mM (1:100), working solution is 4µM (1:25 dilution of intermediate) in 1x PBS. Hoescht (Biotium) was diluted 1:500 and added to the working stain.
Stain Protocol
Murine lens cells were incubated for 2 days at 37°C in 500µL medium 199.
After 2 days, lenses were stained with SynaptoRed™ C2 (1:25, final concentration=4µM) and Hoescht (1:500, 1µL) in 480 µL medium 199 for 5 minutes at room temperature.
Cells were then washed with PBS and imaged immediately in a divot with a 63x oil objective as previously described (1) with 488 nm excitation/598-758 nm emission for SynaptoRed™ C2 (grey) and 405 nm excitation/410-546 nm emission for Hoescht (blue).
Whole murine lens cells stained with SynaptoRed™ C2 and Hoescht to visualize membranes and nuclei, respectively.
Guidelines and Warnings
Tissue collection requires prior approval by the user's Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Protocol references
1. Emin G, Islam ST, King RE, Fowler VM, Cheng C, Parreno J. Whole Mount Imaging to Visualize and Quantify Peripheral Lens Structure, Cell Morphology, and Organization. J Vis Exp. 2024 Jan 19;(203):10.3791/66017. doi: 10.3791/66017. PMID: 38314859; PMCID: PMC11623403.