Formaldehyde

Samples were prepared for SEM by two methods: lyophilization (to visualize the pores) and glutaraldehyde fixation (to visualize the surface). For lyophilized hydrogels, the samples were first frozen at −80 °C for 2 days which were then freeze‐dried for 3 days. The dried samples were sliced and 5 nm of gold was sputter‐coated on the cross‐section to visualize the pores using a SEM (JEOL). To view the surface topography of the microwells, the micropatterned hydrogels were fixed with a fixative containing 3.0% formaldehyde (Electron Microscopy Sciences) and 1.5% glutaraldehyde (Electron Microscopy Sciences) in a 0.1 m NaCacodylate buffer (pH 7.4), at room temperature for 1 h. The samples were then washed and post‐fixed in 1% osmium tetraoxide (Sigma‐Aldrich) on ice for 1 h. These samples were then dehydrated using a graded series of ethanol (50%, 70%, 95%, 100%) and three 15 min washes in fresh 100% ethanol at room temperature. The ethanol was then replaced with a graded series of hexamethyldisilazane (HMDS, Electron Microscopy Sciences) (50%, 75%, and 100%) followed by two 30 min washes in fresh 100% HMDS. After the HMDS wash, the HMDS was allowed to evaporate at room temperature to completely dry the samples. These dried samples were sputter‐coated with gold and the microwells were visualized at an angle using a SEM (JEOL).

In total, 100 µl of SF or EVF were mixed with 400 μl fixative containing 2.5% formaldehyde and 2.5% glutaraldehyde (Electron Microscopy Sciences). PBS was exchanged with the fixative solution using an Amicon‐0.5 ml device with a 3 kDa cut‐off filter and concentrated to 100 μl. After 45 min of fixation, a 200‐mesh formvar and carbon-coated grid (Electron Microscopy Sciences) was placed on top of a 7 μl droplet of SF and EVF samples for 20 min. Next, the grid was transferred 7 times onto drops of water for 2 min. Negative staining was performed with 4% uranyl acetate for 12 min. Grids were imaged using a JEOL JEM‐1010 electron microscope equipped with an AMT digital camera (JEOL, Ltd.). Sample preparation and analysis were performed in triplicate.

Tissues were excised, washed in PBS, cut along the mesenteric plane, pinned flat, and then fixed in 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield PA) and 2.5% formaldehyde (Electron Microscopy Sciences), in 0.1M cacodylate buffer pH 7.4 containing 0.1 mM EGTA for 10 minutes at RT with gentle flushing. The tissue was then cut into small pieces, and fixed for an additional 1 hour in the same fixative at RT. Tissues were washed with 0.1 M cacodylate buffer, and then loaded into a planchette (Technotrade International, Manchester, NH) with PBS containing 20% BSA and 5% FBS, and subjected to high pressure freezing using a Wohlwend High Pressure Freezer (Technotrade International). Rapid freeze substitution, as described [79 (link)], was done using 1% osmium tetroxide, 0.5 % uranyl acetate, 95% acetone and 5% dH2O. After freeze substitution, the tissue was infiltrated with graded acetone into LX112 resin (Ted Pella, Inc. Redding ,CA). Ultrathin sections were cut with a Leica Ultracut E ultramicrotome (Leica Microsystems, Wetzlar Germany), placed on formvar and carbon coated grids, and then stained with 2% uranyl acetate (Electron Microscopy Sciences) and lead citrate (Sigma-Aldrich). Grids from each treatment were imaged using a JEOL 1400 electron microscope (JEOL USA, Peabody, MA) equipped with an Orius SC1000 digital CCD camera (Gatan, Pleasanton, CA).

Immunofluorescence staining of HUVECs was performed after simultaneous fixation and extraction with the mixture of 4% formaldehyde (#15710; Electron Microscopy Sciences) and 0.3% Triton X-100 in PBS for 15 min. After washing with PEM buffer, extracted unfixed cells were incubated with the primary antibody, washed with the PEM buffer, and fixed with 0.2% glutaraldehyde. After quenching with 2 mg/ml NaBH4 in PBS for 10 min, cells were stained with Alexa Fluor 488 phalloidin (Invitrogen, # A12379, 1:200) and Alexa Fluor 594 anti-rabbit IgG antibody (Fisher Scientific, # A11037, 1:100). Coverslips were mounted to slides with ProLong Gold antifade mountant (#P36941; Molecular Probes). Light microscopy was performed using an Eclipse TE2000-U inverted microscope (Nikon) equipped with Plan Apochromat 20× 0.75-NA and Apochromat 100× 1.3-NA objectives and Cascade 512B CCD camera (Photometrics) driven by MetaMorph imaging software (Molecular Devices).

For NOGO-B/RTN4B labelling, cells were fixed with −20 °C methanol, blocked with 10% goat serum (Gibco-Life Technologies) and 1% BSA (bovine serum albumin), labelled with indicated antibodies and mounted in Mowiol (Hoechst, Frankfurt, Germany) supplemented with Dabco (Sigma-Aldrich). All the other samples were fixed with 4% formaldehyde (Electron Microscopy Sciences, Hatfield, PA), 0.1 mM MgCl₂, and 0.1 mM CaCl₂ in phosphate-buffered saline (PBS), quenched with 50 mM NH₄Cl, permeabilized with 0.1% Triton X-100, and then blocked with 0.2% BSA in Dulbecco PBS. When appropriate, the cells were then incubated consecutively with primary and secondary antibodies, diluted in blocking solution. Samples were mounted in Mowiol supplemented with Dabco. Western blotting was done with indicated antibodies according to manufacturer’s instructions and by using standard protocols.