Thermal mixer

hHOs were transferred to microcentrifuge tubes (Eppendorf) using a cut 1000 μL pipette tip to avoid disruption to the organoids and fixed in 4% paraformaldehyde solution. Fixation was followed by washes in PBS-Glycine (20 mM) and incubation in blocking/permeabilization solution containing 10% Donkey Normal Serum, 0.5% Triton X-100, 0.5% bovine serum albumin (BSA) in PBS on a thermal mixer (Thermo Scientific) at minimum speed at 4 °C overnight. hHOs were then washed 3 times in PBS and incubated with primary antibodies (Suppl. Table 1) in Antibody Solution (1% Donkey Normal Serum, 0.5% Triton X-100, 0.5% BSA in PBS) on a thermal mixer at minimum speed at 4 °C for 24 h. Primary antibody exposure was followed by 3 washes in PBS and incubation with secondary antibodies (Supplementary Table 1) in Antibody Solution on a thermal mixer at minimum speed at 4 °C for 24 h in the dark. T-tubules staining was conducted using Wheat Germ Agglutinin (WGA) lectins conjugated with FITC (Millipore Sigma). The stained hHOs were washed 3 times in PBS before being mounted on glass microscope slides (Fisher Scientific) using Vectashield Vibrance Antifade Mounting Medium (Vector Laboratories). 90 µm Polybead Microspheres (Polyscience, Inc.) were placed between the slide and the coverslip (No. 1.5) to preserve the 3D structure of the organoids.

Supernatants with unbound proteins were removed and the beads re-suspended in bead wash buffer (1 mL). The beads were washed twice with bead wash buffer (1 mL) then transferred to clean 1.5 mL microcentrifuge tubes (ThermoFisher Scientific, Waltham, MA). After washing the beads with PBS (1 mL), hFXN-M and mFXN-M were eluted with 200 μL of elution buffer (100 mM acetic acid in 10% ACN) by shaking the beads in a thermal mixer (ThermoFisher Scientific, Waltham, MA) at 1000 rpm and RT for 1 h. Supernatants were then transferred to Sarstedt 2.0 mL LB microtubes and dried under a nitrogen flow using an N-Evap concentrator (Organomation, Berlin, MA).