Feb 06, 2025
Refractive Index Matching - Ethyl Cinnamate
- 1Allen Institute for Neural Dynamics;
- 2Boston University
- Allen Institute for Neural Dynamics
Protocol Citation: Holly Myers, Daphne Toglia 2025. Refractive Index Matching - Ethyl Cinnamate. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj8k4bgk5/v1
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: May 23, 2023
Last Modified: February 07, 2025
Protocol Integer ID: 82287
Keywords: Refractive index matching, Whole mouse brain delipidation, SmartSPIM imaging
Abstract
This protocol details index matching for delipidated whole mouse brain samples in ethyl cinnamate solution to prepare for lightsheet imaging. Upon completion of this protocol, the delipidated mouse brain should be a light amber color and appear translucent when immersed in ethyl cinnamate. This index matching protocol may be paired with a modified iDISCO delipidation protocol: Whole Mouse Brain Delipidation - Dichloromethane. Ethyl cinnamate is an inexpensive, effective, and lower-toxicity index matching option that may be used in place of index matching reagents typically paired with this delipidation protocol (Hildebrand et al, 2019).
Materials
Reagents:
Ethyl cinnamateMerck MilliporeSigma (Sigma-Aldrich)Catalog #112372
| Materials | Product number | |
| 20mL glass scintillation vial | Millipore Sigma, Z376817-1PAK | |
| Serological pipet | Fisher Scientific, 13-678-11E | |
| Aluminum foil | Amazon, B074NB5CDZ | |
| Spatula | Cole Parmer, UX-06287-07 | |
| Petri dish | ThermoFisher, 150460 |
Safety warnings
Wear gloves at all times during this protocol.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
This protocol is designed to index match a delipidated whole mouse brain specimen. For delipidation methodology, reference protocol Whole Mouse Brain Delipidation - Dichloromethane.
Index Matching with Ethyl Cinnamate
Index Matching with Ethyl Cinnamate
3d
3d
Ethyl Cinnamate is typically stored at 4 °C . Thaw bottle at Room temperature for 04:00:00 ahead of use, or until the reagent is no longer frozen.
4h
Using a serological pipet, measure 10mL of Ethyl Cinnamate into a 20mL glass scintillation vial. Place the delipidated mouse brain specimen into the vial of Ethyl Cinnamate using a spatula.
Protect the vial containing the brain submerged in Ethyl Cinnamate from light by covering the vial with aluminum foil or placing the vial in a box. Leave for 72:00:00 at Room temperature .
3d
Assessing for Successful Index Matching
Assessing for Successful Index Matching
3d
3d
Assess the brain specimen for successful index matching. It may be helpful to temporarily transfer the brain to a clear petri dish and completely immerse in Ethyl Cinnamate for this step, using a spatula to gently manipulate the brain in the dish if necessary.
Successful index matching:
When totally immersed in Ethyl Cinnamate, the brain should have an amber color and appear transparent, without visibly opaque interior structures. It should be possible to view numbers, letters, or fine lines on a grid through the brain without warping.
Example of a brain successfully index matched with ethyl cinnamate. Note that when immersed in a dish of ethyl cinnamate, it is possible to view the gridlines and the number "29" through the brain. This brain is ready to be imaged.
Unsuccessful index matching:
Parts of the brain around the cortex may appear transparent, while the deeper structures may have an opaque appearance. It will be difficult to view letters, numbers, or other images through the brain.
Example of a brain not successfully index matched. Note that brain is still opaque and you cannot view any gridlines or numbers through this brain when it is immersed in ethyl cinnamate. This brain should be returned to ethyl cinnamate for longer index matching.
If the brain appears successfully index matched, it is now ready for light sheet imaging. After imaging, the brain may be stored in Ethyl Cinnamate for several months at Room temperature .
If the specimen isn’t properly index matched, consider changing the Ethyl Cinnamate and leaving it for another 24:00:00 at Room temperature before imaging. The time required for clearing and dehydrating is dependent on the size of the specimen -- the larger the specimen, the longer it should be washed in each reagent in order to allow the reagent to fully penetrate the tissue.
1d