Uc7 microtome

Hearts from 6-month old WT (n = 3), 6-month old heterozygous siblings (n = 3), 72-h old WT (n = 4) and 72-h old WT heterozygous siblings (n = 4) were dissected out. Tissues from adults were separated into atrium and ventricle (12 samples in total). The samples were fixed in 2% v/v glutaraldehyde in 0.05 m sodium phosphate buffer (pH 7.4) for 24 h. Following fixation, samples were briefly rinsed in a sodium cacodylate buffer (0.15 m, pH 7.4). A 2 h post-fixation step followed (1% w/v OsO4, 0.05 m potassium ferricyanide in 0.12 m sodium cacodylate buffer, pH 7.4). Following the post-fixation incubation, samples were dehydrated in ethanol and transferred to propylene oxide, before embedding in Epon. Sections were cut at approximately 80 nm on a Leica UC7 microtome. Sections were collected on copper grids in Formvar supporting membranes, and stained with uranyl acetate and lead citrate. Samples were imaged on a Philips CM 100 TEM (Philips, Eindhoven, The Netherlands), operated at an accelerating voltage of 80 kV. Images were captured with an OSIS Veleta digital slow scan 2k × 2k CCD camera, and subsequently viewed in the ITEM software package.

We followed a standard sample preparation protocol for in-resin CLEM (Kukulski et al., 2012 (link)). Immediately after barcoding, the yeast biomass was collected using a Millipore filtering setup on a 0.45-µm nitrocellulose filter. The cell slurry was transferred to the 0.1-mm-deep cavity of a 0.1/0.2-mm membrane carrier for an HPM010 high-pressure freezing machine or Leica ICE. The cavity was covered by the flat side of a 0.3-mm carrier, and the sandwich was inserted in the high-pressure freezing machine. Resin embedding was performed using a Leica AFS2 freeze-substitution machine equipped with a processing robot. Samples were embedded in Lowicryl HM20 resin using the freeze-substitution and embedding protocol optimized for in-resin CLEM (Kukulski et al., 2012 (link)). Dry acetone with 0.1% uranyl acetate was used as the freeze-substitution medium. The blocks were trimmed with a razor blade, and 100-nm-thick sections were produced using a Diatome 35° knife on a Leica Ultracut UCT or UC7 microtome. The sections were mounted on 200 mesh copper grids with continuous carbon support film (Electron Microscopy Sciences). Grids were imaged under the fluorescence microscope the same day.

Pupal and adult IFM samples were fixed at room temperature (RT) in 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (PB) for at least 2 hr and were then transferred to 4°C overnight. On the next day, samples were fixed with 2% OsO₄ in 0.1 M PB for 1 hr on ice in the dark. Next, samples were washed five times using ddH₂O and stained en bloc with 2% uranyl acetate (UA) for 30 min at RT in the dark. After five washes in ddH₂O, samples were dehydrated in an ethanol series (50%, 70%, 80%, 90%, 95%), each for 3 min on ice and twice in 100% ethanol for 5 min at RT. Following dehydration, samples were incubated twice in pure propylene oxide (PO) for 15 min and then transferred to an epon-PO mixture (1:1) to allow resin penetration over night. After removal of PO by slow evaporation over 24 hr, samples were embedded in freshly prepared epon (polymerization at 60°C for 24 hr). 90 nm sections were prepared on a Leica UC7 microtome and stained with 2% UA for 30 min and 0.4% lead citrate for 3 min to enhance contrast. Images were acquired with a Zeiss EM900 (80 kV) using a side-mounted camera (Osis).

Samples were high-pressure frozen followed by freeze-substitution as described previously (Kolotuev et al., 2009 (link)) and flat embedded, targeted and sectioned using the positional correlation and tight trimming approach (Kolotuev, 2014 (link)). For ultramicrotome sectioning, a Leica UC7 microtome was used. 100 nm sections were collected on the slot-formvar coated grids and observed using a JEOL JEM 1400 TEM microscope (JEOL, Japan). Samples were aligned and rendered using the ImageJ and IMOD programs.

The flies were staged and dissected as described in [47 (link)], with the modification that 0.1 M phosphate buffer (PB), pH 7.0, was used as the dissection solution. After dissection, samples were fixed at RT in 4% PFA and 0.5% glutaraldehyde in 0.1 M PB for 3 h. The samples were transferred to 4 °C ON. On the next day, samples were fixed in 2% OsO₄ in 0.1 M PB for 1 h on ice in the dark. Samples were washed 5 times with ddH₂O and stained en bloc with 2% uranyl acetate (UA) at RT for 30 min in the dark. After another washing step (5 times ddH₂O), the samples were dehydrated in a graded ethanol series (30%, 70%, 80%, 90%, 95%) each on ice for 3 min and 2 times 100% ethanol at RT for 5 min. Following dehydration, samples were incubated in pure propylene oxide (PO) 2 times for 15 min and then transferred to an epon PO mixture with a ratio of 1:1 ON to allow resin penetration. After the PO was removed by slow evaporation over 24 h, samples were embedded into freshly prepared epon (polymerization at 60 °C for 24 h). Sections of 90 nm were cut on a Leica UC7 microtome and were additionally stained with 2% UA for 30 min and 0.4% lead citrate for 3 min to enhance the contrast. Images were acquired with a Zeiss EM 900 (80 kV) using a side-mounted camera from Osis.