Timing: 1 day

In this section, we explain the protocol used to conjugate PFFs to 5 nm nanogold beads (Cytodiagnostics). We altered and optimized the standard conjugation protocol provided by the manufacturer (see product sheet on the manufacturer’s website: https://www.cytodiagnostics.com/collections/NHS-Activated-Gold-Nanoparticles).

Note: Since PFFs are very susceptible to aggregation, the nanogold-PFFs need to be administered to cells immediately after conjugation. Prepare the cells such that they are ready to coincide with the conjugation of PFFs to nanogold. Following the administration of PFFs to cells, proceed to prepare grids to characterize the nanogold-PFFs.

Note: all of step 1 should be performed in a tissue culture hood.

The conjugation of PFFs with 5 nm nanogold.

Add 48 μL of 5 mg/mL PFFs to 60 μL of the reaction buffer provided with the nanogold conjugation kit.

Thaw reaction buffer for 1 h on ice.

Retrieve 48 μL of 5 mg/mL PFFs (stored at −80°C) and immediately add 60 μL of ice-cold reaction buffer to the aliquot.

Incubate for 2 min at room temperature.

Slowly pipette the mixture up and down.

Add the reaction buffer-PFFs mixture to 1 vial of 5 nm nanogold.

Incubate for 1 h at 18°C.

Add 10 μL of quencher buffer (provided by the manufacturer) to the mixture.

Slowly pipette up and down using a p10 pipette set at 10 μL.

Avoid creating bubbles in the solution.

Incubate at room temperature for 5 min.

Note: the amount of reaction buffer and quencher buffer used, the amount and concentration of protein used, are all specified by the manufacturer (Cytodiagnostics). The steps above are an adaptation of their protocol for our specific conjugation assay.

Note: for PFFs visualization (labeled with nanogold beads, use a magnification between ×30,000 and ×68,000).

CRITICAL: Avoid rapid pipetting of the samples or sudden movements of the experimental microtube as they will aid in protein aggregation.

Characterization of conjugated PFFs.

Prepare a diluted sample of PFFs with a 1 mg/mL concentration.

Dilute using ddH2O.

Place one drop of diluted PFFs on a carbon/formvar covered copper grid and allow the sample to sit for 10 min at room temperature.

Using a Whatman filter paper, remove the drop from the grid.

Fix the PFFs sample on the grid using 2% PFA (retrieved immediately from storage at 4°C).

Place one drop onto the grid and let the sample sit for 10 min at room temperature.

Remove PFA drop using filter paper.

Add one drop of ddH2O onto the grid for 1 min at room temperature.

Remove with filter paper.

Repeat once more.

Stain the grid with Uranyless.

Centrifuge 1 mL of Uranlyess at 14,000 × g.

add one drop of the supernatant to the grid and wait for 5 min at room temperature.

Wash grid with ddH2O as detailed in d.

Remove the drop using a Whatman filter paper.

Let the grid sit overnight on parafilm at room temperature.

CRITICAL: place grids in a box/chamber for their storage overnight to avoid the addition of contaminants (such as bacteria, dust, etc.) to the EM grid.

Visualize using EM to assess and measure the length of fibrils.

See Figure 2 for an example:

Electron micrograph of nanogold conjugated PFFs

Uranyless was used for staining. Arrowheads point to a few examples of conjugated nanogold beads. Arrows indicate optimal examples of nanogold-labeled PFFs: small size and the appropriate amount of conjugation (>1 and <20 beads per PFFs). Scale bar = 100 nm.

Criteria for successful/ideal PFFs conjugation:

The majority of PFFs should have a length of less than 100 nm.

Most PFFs should be conjugated with at least one nanogold bead.

Only 1 in 20 nanogold beads should be unconjugated (free-floating).

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