Index Matching

Background Information

At this step, the sample is rendered optically transparent to minimize light scattering during imaging. This is done using a solution (EasyIndex) with a refractive index (RI) equal to the RI of the proteins in the tissue. When this high RI solution diffuses into the tissue, the sample becomes optically transparent.

Reagents / Equipment Required:

EasyIndex – RI = 1.52 – stored at RT in sealed container
- For this protocol, it is necessary to use the 1.52 RI version of EasyIndex.
- Here are images of 2 representative mouse brains index matched with 1.46 and 1.52 RI versions respectively which highlights the need for 1.52 RI.
Light pad

Protocols

[Note] If a sample will not be imaged until a later date, store the sample in PBS with 0.02% sodium azide.

1) Carefully invert the bottle of EasyIndex a few times to homogenize the solution.

2) Incubate the tissue in 50% vol EasyIndex + 50% vol distilled water shaking at 37°C.

[Note] If the antibodies were not fixed with PFA, incubate at room temperature instead.

[Note] It is important to incubate in a sealed container to prevent evaporation. Perform in the dark or cover any tubes with aluminum foil to protect from light.

Use the following volumes and recommended incubation times:

3) Incubate the tissue in 100% EasyIndex at 37°C (or RT for unfixed antibodies) for the same duration or until transparent.

After index matching, the sample should be clear enough to easily see through while submerged in EasyIndex. If the solution surrounding the sample seems inhomogeneous, it suggests that the sample has not yet been fully equilibrated with the solution and should be incubated further, or that the sample is not fully delipidated. If it is not fully delipidated, simply wash out EasyIndex with PBS and delipidate the sample more.

To evaluate the index matching of the tissue, we recommend using a transparency with a grid on it (see the above images). Place your sample onto a petri dish, immerse it in EasyIndex, and place the gridded transparency underneath. We often use a light pad to illuminate from the bottom for better viewing. If the sample is well delipidated and index matched, the lines (viewed through the tissue) will be straight. Areas that were not delipidated or index matched as well may distort/ warp the gridded pattern showing through the tissue or obstruct the grid lines entirely.

[Note] We strongly advise against reusing EasyIndex as its Refractive Index (RI) changes after the first usage.

[Note] If a sample has been index matched and needs to be recovered and saved or reprocessed, the sample should be washed in PBS at RT with gentle shaking overnight and stored appropriately. You can also store samples in EasyIndex at RT but be aware that they can take on a more yellow color over time that does not affect imaging.

Continue on to mounting and imaging.

Mounting and Imaging