Equine spleen

Our larval injection protocol was adapted from a procedure developed for Tribolium larvae^{87 (link)}. We collected 50 early L2 (24-26 hr after egg deposition) of PG >FLP;Evi5^FRT, PG>Fer1HCH^IR and control animals. Larvae were washed 3X in PBS and dried immediately on a paper towel. Larvae were aligned on double-sided sticky tape that had been glued to a glass slide. The larvae were covered with halocarbon oil (Sigma, #H8898), and liquids were injected into the T3 or A1 segments of the larvae. The following compounds/concentrations were injected: 250 µM FAC, 5 pM holo-Ferritin (Equine spleen, Sigma, #F4503), 5 pM apo-ferritin (Equine spleen, Sigma, #A3660) and 5 pM denatured holo-Ferritin. Denatured holo-Ferritin was generated via boiling holo-Ferritin (Equine spleen, Sigma, #F4503) for 30 min at 95 °C. The holo-Ferritin and apo-ferritin were diluted in PBS buffer.
All injections were carried out with the microinjector apparatus (Tritech Research) at 5 PSI and 0.2 s intervals. Injection volume was 1 μl.

The ferritins from equine spleen (Sigma-Aldrich) were used for TEM characterization. Specifically, 1 μL of ferritin solution diluted 100 times from the original solution was loaded onto the membrane surface at room temperature, followed by drying naturally in the air. At room temperature, TEM characterizations and the EDX mapping of ferritins were carried out using a FEI 80–300 Environmental Titan operated in monochromated mode at 300 kV.
For the characterization at cryogenic temperature, the as-prepared graphene grid with ferritin was plunged-frozen into liquid nitrogen and then carefully mounted onto a TEM cryo-holder (Gatan 626 cryo-holder) using a cryo-transfer station to ensure the entire process under liquid N₂. The cryo-holder is then inserted into the TEM column (~1 s), and during the whole process the sample is kept at −178 °C. TEM characterizations were carried out using a FEI Titan 80-300 environmental (scanning) transmission electron microscope (E(S)TEM) operated at 300 kV. The microscope was equipped with an aberration corrector in the image-forming (objective) lens, which was tuned before each sample analysis.