Normally, in passive labeling, high-copy-number target proteins can deplete unbound antibody from the incubation solution, stalling progress on extending staining deeper into the tissue.
When binding is switched OFF during the beginning of the experiment, antibodies are driven into the tissue instead of immediately binding to target molecules on the tissue’s edge. Then when the antibodies are uniformly dispersed, the buffer conditions change and the antibodies can then bind to their targets.
For many targets, labeling a modest proportion of the proteins is sufficient to extract molecular and morphological details of the cells, thereby revealing their phenotype.This is preferable to achieving ‘saturated’ labeling of superficial tissue sites along with weak (or non-existent) labeling of deeper structures. Similarly, if truly saturated labeling of the entire sample could be achieved passively at great expense of reagents and time, it would yield little additional meaningful information vs. SmartLabel’s approach.
Yes, buffer composition is (currently) proprietary, and use of these buffers is needed to ensure that active labeling performs as intended. Additionally, the warranty on LifeCanvas equipment is only in effect if LifeCanvas consumables are used.
For a cell-type marker like parvalbumin/PV, a marker of a class of inhibitory interneurons that comprise roughly 10% of all neurons, 5-8µl of a 1mg/ml antibody can be used to label an entire mouse brain hemisphere.
No, protocols are designed to enable close to 100% of the antibodies present in the solution to bind to targets in the tissue. The Sample Buffer and Labeling Buffer cannot be reused because their chemical properties have changed over the course of the experiment (which allows efficient labeling). Starting a new experiment with the sample buffer simply will not work as the initial properties (deoxycholate concentration and pH) are vital to the labeling experiment.
Yes, we have protocols that are easily adapted to different sample types and staining goals. We are also working with a small group of lead users via our Early Adopter Program to develop additional labeling protocols for even more diverse applications.
Full Pipeline Protocol – see the “Immunolabeling with SmartLabel” section beginning on page 9
It can work with both primary and secondary staining in addition to dye-conjugated primaries.
It works with both monoclonal and polyclonal antibodies. Similar to other forms of labeling, there are pros and cons to using monoclonal or polyclonal. Compared to polyclonal antibodies, monoclonal antibodies are expensive, require a higher level of skill and more time to develop hybridomas, have a large number of specific antibodies, recognize one epitope, and have low batch-to-batch variability. We recommend using Fab-fragment secondary antibodies, as their single binding domains help ensure that antibodies do not become aggregated (resulting in poor staining).
You can stain 3 different targets at the same time if you have a 3 channel laser (4 if you have a 4 channel laser). The detergent deoxycholate prevents the primary and secondary antibodies from forming aggregates.
Yes, small fluorescent dyes like SYTO16 (488-excitable) and TOPRO3 (642-excitable) work very well. Cell-type markers expressed in the cell body, such as neuronal markers like NeuN, GABAergic interneuron subtype markers like parvalbumin (PV), calbindin (CB), and calretinin (CR), and glial cell markers like the astrocyte marker GFAP, all work very well.
The tissue needs to fit comfortably inside the boundaries of the 12.6 mm diameter (small) or 19 mm diameter (large) sample cup. If it can fit inside the membrane, it is possible to stain samples larger than mouse organs, such as rat organs or human samples.
In theory, yes, it is possible. We will provide protocols for each antibody optimized for SHIELD, so you will get the best results using our system through SHIELD.
LifeCanvas products have been designed and optimized with the SDS-based techniques, and now with SHIELD, in mind. We have successfully imaged iDISCO-processed samples on our SmartSPIM microscope. We have yet to attempt SmartLabel processing on this type of sample, but since there is only a partial delipidation with iDISCO, immunolabeling may not be sped up to the degree evident for SDS-processed samples. It is also hard to predict how iDISCO-processed tissue would respond to being taken out of the organic solvents and getting rehydrated in our aqueous buffers. Without the support of CLARITY’s hydrogel or a strong fixation like in SWITCH or SHIELD, maintaining structural integrity of the samples may be challenging.
Organs that clear well (such as fleshy ones like liver, kidney, intestine, pancreas) work best. The majority of our work is done with mouse and rat brains.
This has yet to be thoroughly explored. Theoretically it could, but to image a sample of that size it must be cleared anyway, so it is best to delipidate the sample before labeling with SmartLabel.
SmartLabel’s immunostaining can indeed be completed within 24 hours for a mouse brain hemisphere.
After labeling, the sample just needs to be suitably refractive index matched before it can be imaged. Our in-house workflow includes 2 rounds of RI matching, both before and after the sample is mounted within an agarose gel. It is however possible to proceed towards imaging more quickly by incubating the sample at 37°C in RI matching solution such as our EasyIndex for just a few hours, depending on mounting strategy, sample size, and imaging goals.
Please contact the LifeCanvas team at email@example.com. We may be able to point you to another antibody for the same target which is already validated.
For validated antibodies we have a recommended amount determined empirically. In general, the amount of antibody required can be estimated by considering the abundance of antigenic sites in the tissue as a function of the sample’s size.
See the Antibody Cocktail Preparation section of the SmartLabel Protocol (page 24). There is an additional Excel Secondary Antibody Volume Calculator [.xlsx] available for download that will allow you to easily calculate the ideal amount of secondary antibody.
Use of too much antibody can lead to higher levels of non-specific staining and a high background.
The sample cup insert is a mesh bag with a flat bottom. The sample is loaded into this insert which then goes into the sample cup. This mesh bag has holes in the bottom near the corners. These holes allow for more efficient mixing of the sample buffer and antibodies around the sample.
To keep the samples aligned vertically in the cup, you can fold or roll up some nylon mesh and insert that in the cup with the sample to hold it in place. A simple diagram like this one can be found in the protocol:
Store the sample cups in a sealed container filled with a solution containing 5% SDS, 50-100 mM Tris, and 0.02% sodium azide at room temperature. This solution will wash off any remaining deoxycholate and will prevent any remaining antibodies from sticking to the membrane.
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