For early-stage large blastomeres, the lipophilic dye can be applied to the cell by pressure microinjection. Dye is dissolved in vegetable oil (or any type of non-toxic oil that doesn't solidify in the operating and storage temperature) and is back-loaded into injection micropipette. Beware that oil moves much slower, it would take hours or days for the backloaded oil to reach the tip. So, it is recommended that the dye-oil mixture is loaded at least one day before labeling. Before injection, use Kim-wipe to clean the exterior of the micropipette (of course, don't touch the tip) to remove oil remaining on the outer surface of the micropipette. When injecting, first, stab the micropipette into the cell as a regular injection, but do not start applying pressure right away. Retract the micropipette slowly and only apply pressure immediately before the tip leaves the cell surface. Ideally, we want to place a small oil droplet right beneath the plasma membrane so the content of the droplet would soon integrate into the membrane.

For labeling cell(s) in late embryos, it is too difficult to control the location of oil droplet when using pressure injection. However, microinjection is the only way to reach the deeper cells. On the other hand, we had succeeded in using NeuroTrace Tissue Labeling Paste (Molecular Probes N22880, N22881, N22883, N22884) to label superficial cells. Minuten pin glued to a yellow tip was fixed on a stick. Simply dip the tip of the pin in the labeling paste to load the dye. The dye can be applied to a patch of cells (or single cell) by brief contact with the paste on the tip of the pin. In principle, this can be done free-hand. However, few has the hand stability for the task. One can mount the pin to a micromanipulator to improve stability.

Recipes

NeuroTrace Tissue Labeling Pastes are ready to use. Four variants are available:

N22884 is a multicolor kit, containing DiI (red; Em= 565 nm; N22880), DiO (green; Em= 501 nm; N22881), DiD (far red; Em= 665 nm; N22882); DiD paste is currently not available individually. N22883 is CM-DiI paste which in not included in the kit; CM-DiI survives better than regular DiI in the fixed and permeabilized samples.

To prepare the dye-oil mixture, first, dissolve the DiI (or other spectral variants) crystal in EtOH at 100 mg/mL. Then, mix the ETOH stock with oil 1:4. Allow EtOH to evaporate completely (after settle down, EtOH layers on top of oil), the final concentration of dye in oil would be 25 mg/mL. A lower concentration of dye can be used. We found 1 mg/mL dye in oil works just fine.

Available variants are:

• DiO (D275)
• DiI (D282)
• DiD (D307)
• DiR (infrared; Em= 780 nm; D12731)

• DiIC12(3) (D383)
• DiIC16(3) (D384)
• DiOC16(3) (D1125)

These short-chain variants are less hydrophobic and are better for labeling by adding to medium, but this is not necessarily a good thing for direct-contact labeling.

• FAST DiI (D3899)
• FAST DiO (D3898)

These variants migrate faster in the membrane.

• CM-DiI (C7000)
• FM-DiI (F6999)
• SP-DiIC18(3) (D7777)
• DiIC18(3)-DS (D7776)
• SP-DiOC18(3) (D7778)
• DiIC18(5)-DS (D12730)

These variants contain additional chemical group to allow fixation and other histological processing.

A sampler kit (L7781) is available.