Nuclear Dye Labeling

Suggested Dyes

We have validated a number of nuclear dyes to cover multiple channels using several different protocols. Many nuclear dyes have different binding kinetics, and therefore may experience penetration problems when labeling. The dyes below are the ones that have worked the best for us and that we recommend, based on their excitation wavelength. Please be aware of the bolded notes as these are very important considerations. 

• 405 nm: DAPI (ThermoFisher D1306) 

  • Note: 405 nm excitation is not recommended for samples larger than ~1.5 mm due to light scattering. 

• 445 nm: DCS2 (AAT Bioquest 17547) 

  • Note: This dye will wash out during electrophoretic labeling and during PFA fixation. So it must be labeled passively after any electrophoretic labeling is completed. 

• 488 nm: Syto16 (ThermoFisher S7578) or Yo-Pro-1 (Biotium 40089)

• 561 nm: Propidium Iodide (ThermoFisher P3566)

• 640 nm: NucSpot 680/700 (Biotium 41035)
  Note: NucSpot 650/655 works as well. However, it can bleed into the 561 nm channel so we don't recommend it. 

• 785 nm: NucSpot 750/780 (Biotium 41038) 

  • Note: This dye can bleed into the 640 nm channel, so do not use it if you need this channel. 

Here are some nuclear dyes that we do not recommend:

• To-Pro-3: This dye can photobleach quite rapidly.

• Sytox Deep Red: This dye binds very strongly and will often give a large gradient. 

• Yo-Yo-1: This dye binds very strongly and will not stain at depth. 

Protocol Options

There are many viable protocols to achieve good labeling with nuclear dyes. The protocol you use will likely be determined by the brightness of the stain you need, convenience, and time. 

Passive Staining

If you will not be labeling your sample with any antibodies, it is very easy to simply incubate your delipidated samples in PBST (0.1% TritonX-100) with added nuclear dyes at 37°C with shaking. This protocol will generally yield the highest signal and it is the simplest but not necessarily the fastest. The duration of the incubation will depend on the size of the sample being labeled. For whole mouse brains, we typically label them using this method in 5 mL of PBST with the recommended amount of dye shown in our Validated List for 3 days at 37°C. This is also the protocol we use when labeling samples with DCS2 because of its unique incompatibilities. 

Active Staining

You can also deliver nuclear dyes along with antibodies during the SmartBatch+ Standard Primary Labeling step, SmartBatch+ Radiant Primary Labeling step, or the SmartLabel Primary Labeling step. However, we have noticed a few things about this:

• The nuclear dyes can adsorb to the surface of the nanoporous membrane in the sample cups. This can contaminate a subsequent labeling run. We recommend getting a separete cup for nuclear dyes. The contamination will wash out with a subsequent labeling run, so if you use that cup for secondary without adding nuclear dye to the liquid, it will be okay to use for primary in a following experiment without contamination. 

• Compared to the passive methods, this usually results in a little bit dimmer staining. 

• If you will be using our Antibody Blocking Solution, we recommend adding the nuclear dye to this step and increasing the duration to 3 days. This will give you nuclear staining comparable to that of the above passive labeling protocol. 

During Passive Delipidation

Some dyes can be added during passive delipidation to combine steps. However, the signal is typically much dimmer in this scenario. 

Applicable dyes: Syto16, Propidium Iodide

Passive Labeling after Delipidation

If you only need to label your sample with nuclear dyes and your sample is not very large, this is a good choice. To do so, add your nuclear dyes to PBST and incubate your samples at 37°C with shaking. For whole mouse brains, 3 days is long enough. 

Applicable dyes: Dapi, Syto16, Yo-Pro-1, Propidium Iodide, NucSpot 680/700, NucSpot 750/780

During Passive Blocking

If you are going to block your samples using our Antibody Blocking Solution before electrophoretic labeling, it is possible to add the nuclear dyes to the blocking solution. In this case, it is best to increase the duration of the blocking to ensure that labeling is uniform. For whole mouse brains, we extend this to 3 days at 37°C. This typically gives nice and bright labeling. We don't recommend this for extremely large samples like rat brains and larger as the duration can be too long. 

Applicable dyes: Dapi, Syto16, Yo-Pro-1, Propidium Iodide, NucSpot 680/700, NucSpot 750/780

During Electrophoretic Primary Labeling (Radiant or Standard)

You can add nuclear dyes directly to the sample cup during primary labeling. This is the fastest way to achieve nuclear dye labeling, but the signal can be dimmer than passive methods. Please note that these nuclear dyes do adsorb to the surface of the membranes on the sample cups. So, we recommend getting an extra sample cup to prevent contamination between rounds of labeling. Similarly, a round of secondary after the primary will wash out the dyes and the cup can be used for subsequent labeling without contamination. 

Applicable dyes: Dapi, Syto16, Yo-Pro-1, Propidium Iodide, NucSpot 680/700, NucSpot 750/780

During Electrophoretic Secondary Labeling

We do not recommend adding any dyes to this step as the labeling is not uniform. 

Post Immunolabeling

DCS2 is a special case, in that it will be washed out of the sample with electrophoresis and PFA fixation that is often used during the normal electrophoretic labeling steps. So, this dye requires passive labeling after any electrophoretic labeling of antibodies. For whole mouse brains we recommend incubating with 2 μL of DCS2 in 5 mL of PBST for 3 days at 37°C.