Calbiochem

The model tissue hydrogels investigated are fibrin networks with stiff dextran beads embedded, as described previously [5 (link)]. To prepare fibrin gels, fibrinogen (CalBioChem, EMD Millipore, Billerica, MA USA) was dissolved in T7 buffer (50 mM Tris, 150 mMNaCl, pH 7.4). Thrombin isolated from salmon plasma (seaRun Holdings, Freeport, ME, USA) is diluted in ddH₂O at 1000 units per millimeter. Fibrinogen, thrombin, and CaCl₂ were combined in 1X T7 buffer to yield a final solution of 10mg/ml fibrinogen, 30 mM Ca²⁺ and 2 U ml thrombin.
Fibrin networks embedded with volume-conserving beads were prepared with cross-linked dextran beads (Sephadex G-25 medium, GE Health Sciences), as previously described [5 (link)]. The beads are swollen with ddH20 to accomplish 92% swelling and are approximately 50–150 microns in diameter. Bead concentrations of 1vol% or 20vol% are used for the fiber hydrogels and fiber-bead hydrogels, respectively. To keep the concentration of fibrin, thrombin, and CaCl₂ consistent, the extra volume % from dextran beads in the 20% case is accounted for by reducing the T7 buffer. The solution of fibrin, T7 buffer, CaCl₂, and beads are pipette-mixed before thrombin is added to reduce the effect of sedimentation of the beads. Some of the T7 buffer (~30 mL) is added to the thrombin prior to mixing in the final hydrogel solution to prevent heterogeneous gel formation.

The antibodies against phospho-(p)-(Ser21) glycogen synthase kinase α (GSK3α), total and p(Ser9)-GSK3β, total and p(Ser473)-Akt, were purchased from Cell Signaling Technologies (Danvers, MA, USA). The antibodies against the β chain of the insulin receptor (IR) and the insulin-like growth factor receptor (IGF-1R) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). The antibody against total GSK3α was obtained from UpState (Lake Placid, NY) and the antibody against β-actin was bought from BioLegend (San Diego, CA). The guinea-pig anti-CB₁R antibody was obtained from Frontier Institute co., ltd. (Hokkaido, Japan), and we have tested its selectivity before (Bitencourt et al., 2015 (link)). The chemifluorescence (ECF) reagent was acquired from GE Healthcare (Chalfont St. Giles, UK). 2-deoxy-2-(3-(methyl-3-nitrosoureido)-D-glucopyranose (streptozotocin or STZ) and other unspecified (in)organic reagents were from Merck (formerly, Calbiochem, Sigma-Aldrich, Merck-Millipore Corporation; Darmstadt, Germany).