LifeCanvas Wiki

This document outlines the full LifeCanvas Technologies protocol, from beginning to end. There are many options, allowing for both passive or active (electrophoretic) processing. There are also additional steps which are optimized for particular sample types.

Our protocol is based on these linked publications^1,2,3,4, with modifications we have built upon over time.

If this is your first time using this protocol, please consult the appendices for instructions related to washing your device, leak testing cups, etc. If you are looking for tips on how to schedule this protocol, consult this suggested schedule. This schedule lays out the optimal time that steps can be performed in order to complete the protocol most efficiently while also avoiding steps on weekends and evenings.

6 Main Processing Steps:

Please click the buttons to proceed to each step.

In this step, the animal is perfused to remove blood from the tissue of interest. In this section, we provide tips for improved perfusions as well as the alternate protocol from the publication by Park et al..

In this step, the PFA-fixed samples are preserved with a epoxy-based fixative. This ensures that proteins, nucleic acids, and general sample morphology are optimally preserved during the subsequent steps.

Delipidation refers to the process of dissolving the lipid membranes and other lipids inside the sample. This reduces light scattering and allows for future index matching of the sample. We use an aqueous detergent for this process and are no longer using Sodium Dodecyl Sulfate (SDS). Our new reagent, simply called Delipidation Buffer, does not degrade proteins, and it ensures the sample remains the same size throughout the process. This step can be performed passively by incubating the samples in a solution, or it can be sped up by using a SmartBatch+ or SmartClear II Pro electrophoretic device.

This optional step allows you to add fluorescent dyes or antibodies to the tissue to target and image specific proteins of interest. This step can be performed passively by incubating the sample with the dyes or antibodies. By using SmartBatch+ or SmartLabel eFLASH-based labeling, immunolabeling is sped up and penetration/uniformity can be improved versus passive labeling.

In this step, the refractive index of the sample is homogenized to render the sample transparent. Once the sample is transparent, it is then mounted in an agarose gel and further index matched before imaging.

This process outlines steps and tips for preparing your sample for imaging. This will involve immobilizing your sample and avoiding dust or bubbles that would interrupt the path of light.

We also have some sample-specific protocols for specialized tissue types. We will continuously add to this list in the future:

This is a specialized protocol optimized for processing organoids of various types and sizes.

Samples that exceed SmartBatch+ size limits, SmartSPIM limits, or have penetration issues can also be sliced with our Megatome technologies.

Publications

1. Murray et al., (2015). Cell. 163(6):1500-14.

2. Nudell et al., (2022), Nature Methods. 479–485

3. Park et al., (2019). Nature Biotechnology. 37: 73–83.

4. Yun et al., (2019). bioRxiv