Lead citrate

lead citrate (0.4%) was prepared by taking (0.04 g) of lead citrate (Fluka) in 10 ml of boiled water and immediately vortexed vigorously for proper dissolving of lead citrate. Subsequently, for the complete dissolving of the lead citrate, 100 μl of 10 N NaOH (Sigma) solution was added to the 10 ml lead citrate and the mixture was vortexed till it gets dissolved. Few pellets of NaOH was kept in a clean dried Petri dish and was covered with lid for 30 min to block the air contact. Consequently, a drop of lead citrate solution was added on the parafilm and the dish was closed again for 10 min. Subsequently a drop of 0.1% NaOH solution was added in a separate Petri dish and similarly, one more Petri dish was taken with parafilm and added a drop of autoclaved de-ionized water. The copper grid having the ultra-thin sections was kept inverted on the lead citrate solution, which was prepared earlier and immediately it was transferred to the 0.1% NaOH solution for 10 min. Finally, grids were transferred to the fresh water drop which was kept separately in another Petri dish for 10 min. Both the washing steps mentioned above were repeated twice and finally grids were kept overnight on a Whatman No. 1 filter paper in upright position. Grids were observed under transmission electron microscope at 120 kV (BioTwin, FEI).

For contrasting and carbon coating of the samples, cover glasses were removed and mowiol was washed out with ddH₂O and blow dried. The object glass area containing the ribbon was cut out with a diamond pen (Roth, Karlsruhe, Germany). A 2.5% (w/v) uranyl acetate (Merck, Darmstadt, Germany) in ethanol solution was dropped onto the sections and incubated for 15 min at RT. Sections were briefly washed in 100% ethanol, 50% (v/v) ethanol in ddH₂O, and 100% ddH₂0, followed by 10 min incubation at RT with 50% (v/v) lead citrate solution in decocted H₂O containing 80 mM lead citrate (Merck, Darmstadt, Germany) and 0.12 M trisodium citrate (AppliChem, Darmstadt, Germany) (Reynolds, 1963 (link)). After washing in ddH₂O, sections were dried and attached to specimen pin mounts via carbon conductive tape (Plano, Wetzlar, Germany). Conductive silver (Plano) was applied, connecting the glass with the edges of the holder. A 5 nm carbon coat was applied, using a CCU-010 carbon coating machine (Safematic, Bad Ragaz, Switzerland). Serial sections were imaged in a JSM-7500F field emission scanning electron microscope (SEM; JEOL, Tokyo, Japan) with an acceleration voltage of 5 kV, a probe current of 0.3 nA, and a working distance of 6.0 mm. At each region of interest (ROI), several images with increasing magnification were acquired.

For TEM analysis, samples were fixed in Karnovsky’s Fixative consisting of 8% PFA, 25% glutaraldehyde (Agar Scientific, Stansted, UK), and 0.2 M cacodylate buffer (Agar Scientific) for 1 hour before washing in 0.1 M cacodylate buffer. Samples were further fixed in 1% osmium tetroxide (Agar Scientific) for 1 hour, washed in 0.1 M cacodylate buffer before being dehydrated through an ethanol series before embedding in Epon resin (Agar Scientific) containing dodecenylsuccinic anhydride (DDSA), methyl nadic anhydride (MNA) and benzyldimethylamine (BDMA). Ultra-thin (70 nm) sections were obtained using a diamond knife (Agar Scientific) and loaded onto formvar-coated slot grids (Agar Scientific). Grids were then stained with uranyl acetate (Agar Scientific) and lead citrate (Sigma Aldrich) prior to imaging using a Hitachi H7600 electron microscope.