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. 2020 Aug 18;11:2041731420945633. doi: 10.1177/2041731420945633

Figure 7.

Figure 7.

Immunofluorescence analysis by confocal microscopy for H4-SW cells in 2D, layered, or embedded conditions. For the layered condition, we plated 2.34 × 104 H4-SW cells/cm2 and covered with 1.25- or 2.50-mm-thick gels, prepared by diluting 9 parts (v/v) polymer solutions with 1 part (v/v) culture medium. For the embedded condition, we diluted 9 parts (v/v) polymer solutions with 1 part (v/v) cell suspension (3.125 × 105 H4-SW cells/ cm2) and prepared 2.50-mm-thick samples. We stained cell nuclei with Hoechst 33342 (blue), cell membranes with a lipophilic tracer (red), and soluble amyloid fragments with 6E10 primary antibody (green). (a) Control cells, (b) cells covered by a 1.25-mm or 2.50-mm-thick layer of COLL-HA semi-IPNs, (c) cells covered by a 1.25-mm or 2.50-mm-thick layer of COLL-PEG3350 gels, and (d) cells embedded in 2.50 mm-thick COLL-HA, COLL-PEG2000, or COLL-PEG3350. For (b) and (c), the panel of images on the left represents the projection on the cellular plane of the signal from the secondary antibody detected in the z-axis (200 μm) of the representative volume of hydrogel samples, while the square on the right shows the representative volume of hydrogel samples. Scale bars = 20 μm. For (d), the upper panel shows the complete staining, while in the lower one we omitted the signals from cell membranes and nuclei to highlight the amyloid signal. The z-axis for the representative volume of hydrogel samples was 200 μm.