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Choosing appropriate antibodies and dyes
Choosing appropriate antibodies and dyes
The protocol can accommodate:
staining with dye-conjugated antibodies
simultaneous delivery of primary along with monovalent Fab fragment secondaries
sequential delivery of primary and secondary (can be Fab or IgG)
Please consult the Validated Antibody List for more information as it can take significant time and work to validate and optimize antibodies. We have also found that some antibodies require specific secondary delivery schemes, or are only bright enough to work in certain channels.
Are you using an antibody for the first time?
We recommend first following the Antibody Validation Protocol. If you wish to skip that step, we recommend using a sequential delivery of primary and secondary for the first test as simultaneous Fab fragments can alter the primary antibody binding affinity. It is also recommended to try new antibodies with 1-2 samples first, as batch experiments can require large amounts of antibody.
Additional notes and recommendations:
If you anticipate a signal being dim, we recommend sequential delivery of secondary using whole IgG secondaries in the 647 nm channel.
Avoid antibody host conflicts with primary antibodies.
Avoid excitation/emission wavelength conflicts between secondary antibodies.
If you are delivering a nuclear dye or vasculature stain, please add them during the primary step. In general, we recommend using longer wavelengths (red or far red) for antibodies and saving shorter wavelengths for brighter targets or nuclear dyes.
If you are using a dye, do so during primary labeling. To prevent the cup from being contaminated with the dye, use the same cup during secondary staining and post wash. This pulls any dye that got stuck in the cup during primary labeling back out of the cup.
If you do not use the same cup in secondary labeling, quarantine the cup to use only with other samples being stained with the same dye.
Protocols
Protocols
BLOCKING
BLOCKING
[Note] Blocking may not be needed for some antibodies. This can be determined empirically. In our hands, it is essential for active labeling with some antibodies such as c-fos.
Preparation (2-3 Days)
Make 5 mL of 5% Donkey Serum in Antibody Blocking Solution per sample.
Incubate each sample in a 5 mL tube for 2 days at 37°C with shaking.
If your samples were previously in an incubation jar, we recommend separating the samples for blocking to avoid using too much donkey serum.
If you are labeling rat brains you should increase the volume to 20 mL and the incubation time to 4 days.
[Tip] You may block for up to 3 days. 3-day blocking will avoid steps on the weekend. See the schedule suggestion for more tips.
[Note] If you are going to be using Goat secondaries, add 5% Goat Serum in Antibody Blocking Solution instead.
PRIMARY LABELING
PRIMARY LABELING
There are currently 2 different Primary labeling protocols. To use the Radiant Protocol, your SmartBatch+ must be upgraded with the necessary hardware. Please consult the Validated Antibody List for recommendations on which protocol to use depending on your target of interest. In general, the Radiant Protocol provides increased uniformity of labeling, especially for targets that are highly abundant and widely expressed in the sample. It also provides increased penetration of antibodies in extremely large samples (larger than mouse brain). However, this protocol does take a little bit longer, especially for Batch labeling experiments.
SECONDARY LABELING
SECONDARY LABELING
Once antibodies have been delivered via either the Standard or Radiant Protocol, the protocol for secondary labeling is the same.
After secondary antibody labeling is completed, you can proceed to Index Matching!
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