Tecnai g2 spirit biotwin instrument

MDA-MB-231 and MDA-MB-468 cells were cultured on a 6-well dish at a density of 1 × 10⁶ cells per well. The cells were incubated with Au-spa-CTX and Au-OPEG-CTX NCs for 24 h. After incubation, the medium containing excess NPs was removed and the cells were washed several times with PBS, detached, and centrifuged at 2000 rpm for 5 min. The primary fixation of the cells was carried out with a 0.1 M PBS solution of 2.5% glutaraldehyde. Then, the cells were washed with PBS and post-fixed using a 1.5% aqueous solution of OsO₄ (caution: extremely toxic) for 2 h followed by successively washing with PBS. Then, the cells were dehydrated using increasing concentrations of ethanol (30–100%). Finally, the cell pellets were infiltrated with a mixture of 1 : 1 epoxy resin in 100% ethanol overnight and then left to polymerize at 60 °C for 48 h. Ultrathin sections (∼70 nm) were cut using a diamond knife. The sections were stained with uranyl acetate and lead citrate solutions and imaged using the FEI 120 kV Tecnai G2 Spirit BioTWIN instrument on 120 kV.

Samples were centrifuged at 6,300 × g for 10 min at 4°C to collect bacterial precipitates and washed twice with 0.1 M phosphate buffer (PB) (68.4 mM Na₂HPO₄ , 31.6 mM NaH₂PO₄ [pH 7.2]). The samples were first fixed with 2.5% glutaraldehyde (>6 h) and then washed four times with 0.1 M PB for 15 min each time. The cells were postfixed with 1% OsO₄ for 2 h and were again washed four times with 0.1 M PB for 15 min. The samples were dehydrated by a graded ethanol series (50%, 70%, and 90%) and dehydrated using 90% ethanol–90% acetone (1:1) and then transferred to absolute acetone. The dehydrated specimens were soaked in a 1:1 mixture of acetone and epoxy for 1 h and then in a 1:2 mixture of acetone and epoxy overnight. Finally, specimens were embedded in 100% epoxy, incubated for 7 h at room temperature, and then placed in an oven (65°C) for 48 h to polymerize. Finally, the prepared specimens were sectioned in an UC6 ultramicrotome (Leica, Germany) and stained with TI Blue for 10 min and then lead citrate for 6 min. The specimens were examined using a Tecnai G2 spirit Biotwin instrument (FEI, Hillsboro, OR).