Methanol

Sampling was performed at week 17 to assess mid- to long-term activity of the HPA axis. Hair was removed from the posterior third of the animals’ dorsal side at week 14, allowed to regrow for 3 weeks, and after this hair growth period, new hair was collected from the shaved area and stored at 4 °C. Sampling was performed in a separate room. Hair samples were homogenized and 40 mg of hair were weighed and placed into 5 mL glass tubes. Samples were washed 3 times with 4 mL of undiluted 2-propanol (Sigma, Spain) using an orbital tube mixer (360° rotation at 20 rpm, Minilab Roller, Sigma) for 3 min, followed by centrifugation (2000 g, 10 min) and removal of the supernatant. Residual 2-propanol was evaporated in a dry bath at 50 °C overnight. Then, 1.6 mL of HPLC-grade methanol (Scharlau, Spain) was added and mixed at room temperature by rotation (20 rpm) overnight. After, the methanol was collected into 2 mL Eppendorf safe-lock tubes and dried in a vacuum centrifuge for 2.5 h until the methanol evaporated. We performed three washes with methanol. Dried samples were stored at − 20 °C and resuspended with 1 mL 0.1 M phosphate buffer.

For each digestion experiment, PK (Merck Millipore) was diluted fresh on the day from a glycerol stock to working stock in cell culture grade water. PK working stock concentrations were calculated to digest RT-QuIC reaction products with different amounts of PK. The final digestion volume included 76 μL of sample and 4 μL of a PK working stock solution. Digestions were carried out at 37°C for 1 hour and stopped by placing the tubes on ice. PK activity was irreversibly inhibited by adding Pefabloc SC (Sigma-Aldrich) to 1.25 mM.
Products of PK digestion were methanol precipitated with 10 volumes of -20°C 99.9% pure methanol (Fisher Chemicals). Samples, with added methanol, were thoroughly vortexed and incubated overnight at -20°C. The following day samples were centrifuged for 35 minutes at 18200 x g (Eppendorf 5417R, rotor 06/09 HL128), the supernatant was discarded, and residual methanol evaporated by incubating the open tubes at 100°C in a Class II microbiological safety cabinet. Pellets were resuspended in 42 μL of 0.1%SDS/PBS, half of the volume was moved to a fresh tube and added to an equivalent volume of either LDS4X/2%βMA (reduced samples) or LDS4X with no βMA (not reduced samples).

The undigested P and FE samples underwent a methanol extraction to quantify the polyphenol content to be used as reference values for the release of polyphenols from the digested samples. The starting material (P and FE) was diluted in 70% methanol (Sigma-Aldrich, Darmstadt, Germany) at a concentration of 100 mg/mL (to maximize the measurement accuracy) and placed in an Eppendorf. After centrifugation (Eppendorf MixMate^®, 10 min, 25 $°\mathrm{C}$ , 5000 rpm), the aqueous phase was collected, and the insoluble fraction was further extracted using another 1 mL of 70% methanol by the same method as described above. The supernatants from each extraction were pooled together for each sample; then, the methanol extracts of P and FE, namely P-MetOH and FE-MetOH, were analyzed for their polyphenol contents (−2.2.4) and used as the reference values [28 (link)].