Method details

A spotted ratfish, Hydrolagus colliei (41 cm, male), was collected by Thomas Quinn (University of Washington) on a research cruise in Puget Sound and brought to the Benaroya Research Institute, Seattle, for tissue processing. Additional expired and frozen adult spotted ratfish (males and females) were obtained from Jason Cope (NOAA), Scott Hamilton (Moss Landing Marine Labs, MLML), and Matthew Jew (MLML). All animal work was conducted under approved Institutional Animal Care and Use Committee (IACUC) protocols: IACUC16-014 (Benaroya Research Institute) and AUP18-0001 (University of California, Merced). Euthanasia of the one ratfish specimen was carried out using tricaine methylsulfonate (Western Chemical Inc., #NC0872873) at 1000 mg/L. Expired and frozen adult spotted ratfish (Hydrolagus colliei) specimens were thawed in a refrigerator overnight, and then AoL gel was removed by applying pressure to the pores with gloved fingers or the blunt side of a scalpel, placed in tubes, and stored at -80°C until use.

Paraffin sections of formalin-fixed H. colliei tissues were prepared using standard processing, embedding, and microtomy protocols and stained following protocols described in the study by Phillips et al., 2020, and epifluorescence images were taken using a Leica DMR upright epifluorescent microscope equipped with a SPOT RT Slider cooled 1.4 megapixel color/monochrome CCD camera and an Insight 4 megapixel color CCD camera (Diagnostic Instruments), or a Keyence BZ-X700 microscope workstation equipped with epifluorescence.

To prepare the samples imaged in Figures 2C and 2D, an aliquot of AoL gel from H. colliei was placed in a small centrifuge tube. Chitin-binding domain (CBD) probes (Phillips et al., 2020) were diluted in 1X phosphate buffered saline (PBS) and pipetted gently on top of the gel sample and incubated overnight at 4°C. The next day, the CBD liquid was gently replaced with Milli-Q water to wash the specimen. Gel was pulled from the tube, placed on a slide, and then imaged using a Leica DMP fluorescence microscope with a Qimaging Retiga Exi CCD camera.

Equal volumes of AoL gel and water were combined in a tube and vortexed vigorously. Proteinase K was added to a final concentration of 40 μg/μL and incubated for one hour at 50°C. An aliquot was then added to dialysis tubing with MWCO 12-14 KDa (Fisher #21-152-15), dialyzed overnight in 5 L of water at 4°C, and then stored at -20°C until use. For SDS-PAGE, 30 μL of gel sample was combined with 10 μL of reducing buffer and incubated at 95°C for 5 minutes. 1X running buffer was prepared, and 1% SDS was added. Samples were loaded into precast gels (Sigma #PCG2001-10EA) along with marker (Lonza ProSieve Color Protein Marker #BMA50550). Gel was run at 50 V for 20 minutes, then at 100 V for 50 minutes, and then rinsed with DI water. Coomassie Brilliant Blue stain (Fisher #501035935) was poured over gel and incubated for 5 hours. Destain was then added to gel and swirled overnight before pictures were taken.

Equal volumes of AoL gel and 1X PBS were combined in a tube and vortexed vigorously. Proteinase K was added to the gel sample to a final concentration of 0.1 μg/μL and incubated at 50°C for one hour. Sample was then dialyzed with 3.5-KDa dialysis tubing in 2 L of water for 5 hours.

Equal volumes of AoL gel and buffer (0.1 M Tris + 0.05 M ethylenediaminetetraacetic acid + 0.1% SDS) were combined and proteinase K was added to a final concentration of 40 μg/μL. The solution was incubated at 50°C for an hour and then heated at 60°C for 30 min to denature remaining enzymes. The resulting solution was dialyzed overnight in 5 L of water with 12- to 14-KDa dialysis tubing (Fisher #21-152-15). Lastly, the sample was sonicated on the continuous setting on ice for 20 minutes.

Several milliliters of AoL gel were dialyzed overnight in 3.5-KDa dialysis tubing in 5 L of water at 4°C to remove salts. A thick-walled polycarbonate lidless centrifuge tube was filled with dialyzed gel (∼500 μL) and then spun at ∼300,000-400,000 x g for 16 hours at 4°C with a TLA-120.1 Beckman Coulter rotor in an Optima Max-XP ultracentrifuge. A sample of Proteinase-K-digested gel was put into a centrifuge tube and also spun at ∼300,000-400,000 x g for 16 hours.

SAXS experiments were carried out at Lawrence Berkeley National Laboratory’s (LBNL) Advanced Light Source (ALS) on Beamline 7.3.3. Synchrotron radiation provides a high-intensity, collimated x-ray beam, ideal for studying poorly ordered, dilute isotropic aqueous materials such as biological gels. Previously prepared gel samples were inserted into 1.5-mm quartz x-ray capillaries (Charles Supper Inc.) by gentle centrifugation and mounted in the beam path in a transmission configuration. Capillaries were mounted in a motorized translation stage, allowing precise control of capillary position in a plane perpendicular to the beam direction. A water-filled capillary was included to obtain scattering data for the water background of the gels. A capillary containing silver behenate was included for beam center calibration and determining the sample to detector distance which was 3529.37 mm. To perform the scattering experiments, we used an approximately 300 μm (H) x 700 μm (W) beam at 10 keV and exposed the gels for 2.0 seconds per capillary at several different positions to obtain the most intense scattering pattern for analysis. The scattering patterns were recorded on a Pilatus 2M detector for analysis. The pixel size of the detector was 172 μm².

Data analysis was performed using the Nika and Irena macros in Igor Pro by WaveMetrics. The patterns showed diffuse scattering and rings of approximately uniform intensity (no significant alignment was observed) and were radially integrated to obtain 1D intensity plots as a function of q, the scattering vector. To perform the integration, we selected sectors on the scattering pattern image at various azimuthal angles and angular widths. The intensity plots shown were taken from the integration of sectors at an azimuthal angle of 60 degrees with an angular width of 20 to 40 degrees. The full 2π scattering pattern was not accessible on this particular beamline setup, so sector widths were chosen to produce data with a high signal-to-noise ratio, while optimizing the q range for our material. The Igor wave arithmetic tool was used to subtract the water background.

An AoL gel sample was placed in 3.5-KDa dialysis tubing, sealed on both ends using dialysis clamps, and left in ethanol 200 proof for several hours (in most cases, overnight). Dialysis tubing became very stiff, and little liquid remained inside after ethanol incubation. Often, white precipitate was observed coating the inside of the tube. Dialysis clamps were replaced with twist ties before introduction to the supercritical drying system. Supercritical drying was performed using a Denton Vacuum, Inc supercritical drying system. The chamber was flushed with liquid CO₂ until no ethanol smell was observed in the effluent followed by a 2-hour soak in pressurized liquid CO₂ to allow exchange of ethanol inside the dialysis tubing. Phase change to supercritical CO₂ was achieved by heating the chamber to ∼50°C (reaching pressure of ∼1400 psi). Final drying was achieved by reducing sample chamber pressure, allowing the supercritical CO₂ to transition to the gas phase. Samples were stored under vacuum until imaging.

AoL gel samples were ambiently dried on a freshly cleaved piece of mica attached to an aluminum stub using a piece of double-sided carbon tape. For supercritical dried samples, a piece of double-sided carbon tape was stuck onto aluminum stubs and then pressed onto the inside of the dialysis tubing containing supercritical dried samples. SEM was performed using the Zeiss Gemini500 FEG-SEM at the Imaging and Microscopy Facility of University of California, Merced. Beam landing energy of 500 eV and secondary electron detection were used.

AoL samples were dropped directly onto freshly cleaved sheets of mica and dried ambiently in a covered box for several hours. AFM was performed using a Veeco Innova instrument in tapping mode. Probes with a spring constant of 40 N/m (Tap300Al-G, Budget Sensors) were used to image air-dried samples drop-cast on freshly cleaved mica to obtain topographical, amplitude, and phase images in air. Images were collected at a scan rate of 1 line per second at room temperature.

Samples of dialyzed gel and proteinase-K-treated dialyzed gel were brought to UC Santa Cruz where analyses were performed. The two-terminal devices used in EIS measurements were fabricated on a glass wafer (Figure 5A). Prior to device fabrication, the substrates were cleaned by sequential sonication in acetone, isopropyl alcohol, and water. Then, a 10-nm titanium adhesion layer overlaid with a 100-nm gold was electron-beam evaporated onto the clean substrates through a shadow mask. The dimensions of the paired electrodes were 1 cm wide by 2 cm long with an interelectrode separation of 50 μm. A polydimethylsiloxane (PDMS) well was made with a 4-mm biopsy punch and bonded to the glass wafer to define the geometry of the gel. The devices were completed by drop casting the native H. colliei AoL gel and digested gel directly into the PDMS well, and the resulting films were allowed to dry in air (Figure S5). We hydrated the samples by incubating them in a home-made sealed chamber at 90% relative humidity (RH) at room temperature for 2 hours with D₂O or H₂O. After incubation, Nyquist plots were recorded using Autolab PGSTAT128N between 100 kHz and 0.1 Hz at 10 mV amplitude and analyzed using Nova 2.0 software. Then, we calculated the conductivity (σ) using the following equation: σ = L / R_b A where A is the cross-sectional area given by the width of the contact (4 mm) multiplied by the thickness of the sample as measured with atomic force microscopy (2.7 ± 1.9 μm for ratfish gel, 0.46 ± 0.27 μm for digested ratfish gel), L is the device length or electrode separation (50 μm), and R_b is the value of resistance obtained from the equivalent circuit we mentioned earlier.