Analysis of Cultures

Subsequent to culture, the patterns of cell differentiation within explants can be examined either by immunohistochemical or in situ hybridization techniques (Fig. 2). In either case, explants can be sectioned directly in the gel and then analyzed, or processed for whole-mount labeling. The decision whether to section or process for whole-mount is largely probe-dependent. With a strong

Fig. 1. Schematic diagram of a three-dimensional collagen gel culture. Side view, showing explants positioned at the interface between the collagen bilayer. The explants should lie in the center of the gel and rest on the bottom collagen cushion. The bottom layer of collagen is flat. The top layer of collagen overlays the bottom cushion precisely. Medium is added just to cover the gel.

Fig. 1. Schematic diagram of a three-dimensional collagen gel culture. Side view, showing explants positioned at the interface between the collagen bilayer. The explants should lie in the center of the gel and rest on the bottom collagen cushion. The bottom layer of collagen is flat. The top layer of collagen overlays the bottom cushion precisely. Medium is added just to cover the gel.

probe or antibody, whole-mount labeling techniques work well (Fig. 2C). With a weaker probe or antibody, sectioning is more appropriate (Fig. 2D).

3.5.1. Immunohistochemical Analysis of Cultures

3.5.1.1. Cryostat Sectioning Explants for Immunohistochemical Analysis

1. Remove culture medium, and replace with 0.12 M PB containing 4% paraformaldehyde, so that explants are immersed in the fixative. Leave at 4°C for 2 h, and then replace fixative with 30% sucrose in 0.1 M phosphate buffer for 24-48 h at 4°C.

2. Using blunt forceps, tease around the edges of the collagen bilayer containing the explant until it lifts off the base of the four-well dish. Transfer onto a glass plate. Trim the gel, so that the explant is contained in a square of collagen, approx 0.6 cm2. Freeze this in OCT (19) (TissueTek).

3. Collect 12-20 |im frozen sections (20) onto gelatin-subbed slides or Superfrost Plus slides, and process according to standard techniques.

3.5.1.2. Whole-Mount Immunohistochemical Analysis of Explant Cultures

1. Remove culture medium, and replace with 0.12 M PB containing 4% paraformal-dehyde, so that explants are immersed in the fixative. Leave at 4°C for 2 h.

2. Using blunt forceps, etch around the edges of the collagen containing the explant until it lifts off the base of the four-well dish and transfer to an Eppendorf tube. Process according to standard whole-mount techniques.

3.5.2. In Situ Hybridization of Cultures

3.5.2.1. Sectioning Explants for In Situ Hybridization

1. Remove culture medium, and replace with 0.12 M PB containing 4% paraformal-dehyde so that explants are immersed in the fixative. Leave at 4°C for 2 h.

2. Transfer explants in collagen to 30% sucrose in 0.1 M PB, leave at 4°C overnight, embed in OCT, and process according to standard techniques.

3.5.2.2. Whole-Mount In Situ Analysis of Explant Cultures

1. Remove culture medium, and replace with 0.12 M PB containing 4% paraformal-dehyde so that explants are immersed in the fixative. Leave at 4°C for 2 h.

Fig. 2. Analysis of cell differentiation within neural explants cultured in three-dimensional collagen gels. (A,B) Transverse sections through an embryonic rat, showing expression of sonic hedgehog mRNA (A) and expression of the FP3 antigen (B) by floor plate (fp) cells of the spinal cord. (C,D) Floor plate cells that differentiate within explanted neural tissue in collagen gels can be detected through in situ hybridization techniques (C) or immunolabeling techniques (D). (C) Shows a neural explant that has been cultured and processed by in situ hybridization with antisense probe to sonic hedgehog. Floor plate cells that express the gene extend in a stripe within the explanted neural tissue. (D) Shows a section through a cultured rat neural explant, labeled to detect FP3. The dotted line in (B) marks the edge of the spinal cord, and in (D), marks the edge of the neural tissue.

Fig. 2. Analysis of cell differentiation within neural explants cultured in three-dimensional collagen gels. (A,B) Transverse sections through an embryonic rat, showing expression of sonic hedgehog mRNA (A) and expression of the FP3 antigen (B) by floor plate (fp) cells of the spinal cord. (C,D) Floor plate cells that differentiate within explanted neural tissue in collagen gels can be detected through in situ hybridization techniques (C) or immunolabeling techniques (D). (C) Shows a neural explant that has been cultured and processed by in situ hybridization with antisense probe to sonic hedgehog. Floor plate cells that express the gene extend in a stripe within the explanted neural tissue. (D) Shows a section through a cultured rat neural explant, labeled to detect FP3. The dotted line in (B) marks the edge of the spinal cord, and in (D), marks the edge of the neural tissue.

2. Using blunt forceps, tease the collagen containing the explant off the base of the four-well dish, transfer to a sterile Eppendorf tube, and then process according to standard techniques (21).

3. Explants can subsequently be viewed directly as whole-mounts or can be sectioned. If sectioning, refix in 4% paraformaldehyde 1-2 h at 4°C, then transfer to 30% sucrose, and process as described above. Optimal signal is maintained on thicker sections (20 pm). Mount sections in Glycergel.

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