1. Although dissections can be performed in PBS, tissues isolated from very young embryos survive far better if dissected into an air-buffered culture medium, such as L15-air. It is of utmost importance to keep tissue ice-cold at all stages (with the exception of the enzymatic treatment step) prior to positioning on the collagen bed. Rat or mouse embryos should be removed from the decidua immediately and placed on ice. Never allow embryos or explanted tissue to remain off ice for more than approx 5-10 min. If working to isolate a specific piece of tissue from an embryo, all the other embryos from the litter should remain on ice. When dissecting small portions of tissue, relax and remember to breathe.

2. Collagen is also available commercially (e.g., Vitrogen). However, the gelling properties of collagen isolated from rat tails are somewhat better than those of commercially obtained samples. Furthermore, the currently tested whole-mount in situ hybridization techniques do not appear to work well on commercially obtained collagen.

3. Collagen cannot be sterilized once made. It is therefore critical to prepare it under sterile conditions. Swab instruments well with ethanol, and during the isolation of the collagen, work in an air-filter hood.

4. The optimal time needed for Dispase to work varies according to the age and tissue, and is best assessed empirically. As a rule, the Dispase can be judged to have been effective when sheets of tissue, such as mesoderm and ectoderm, begin to separate. Tissues can be microdissected easily after an acute treatment with Dispase. Thus, material can be more easily dissected after a 5-min incubation in Dispase at 1 mg/mL than after a 10-min incubation at 0.5 mg/mL. Tissues become difficult to dissect after prolonged exposure to the enzyme and die if overexposed. Do not place too many explanted tissues together in a small volume of Dispase—they will become intertwined, sticky, and difficult to separate. Dispase is made up fresh, but the same batch can be used throughout the day.

5. Take care when transferring the tissues from Dispase to cold L15-air: the tissues coming out of Dispase will be relatively warm, i.e., room temperature, and may thus rise to the top of the colder, denser L15-air, and burst at the meniscus. This can be avoided if the tissues are transferred slowly and into a deep amount of L15-air. Small tissues are especially fragile after being incubated in Dispase and must be transferred with care. To prevent the tissue explants sticking to the transfer pipet, use a glass Pasteur pipet or a pulled glass capillary (see Note 12), and do not draw the tissue explants too far up the pipet. Siliconize the glass pipets if necessary. Always use the microscope to visualize the tissues as they are transferred between vials.

6. Treatment with Dispase results in the tissue becoming sticky as cells begin to break down. Further dissection is facilitated by the inclusion of a small quantity of serum into medium to which tissue is transfered (1 drop in 5 mL). Serum inhibits further action of the Dispase. In addition, nonspecific binding by the serum to the tissue results in it becoming less sticky.

7. If stirred too vigorously or too long, tendon proteins other than collagen will start dissolving.

8. The purpose of the 10X DMEM is twofold. First, it provides a nutritious medium, enhancing the condition of the explanted tissue. Second it serves as a color indicator, facilitating titration of the bicarbonate—the solution will turn pink if too much bicarbonate is added.

9. If insufficient bicarbonate is added, the collagen will not set; if too much is added, it will set too quickly and unevenly.

10. The four-well dishes are convenient to use, and in these wells, gels can be covered with as little as 0.4 mL of medium (important for testing growth factors and conditioned medium).

11. The beds must be flat, since explants will tend to slide off a convex bed.

12. Fragile tissues can be manipulated far better using a pulled capillary tube than a Pasteur pipet. When picking up and transferring small amounts of tissue, always work through the stereo-dissecting microscope. When transferring tissue in a pulled capillary tube, allow the tissue to enter the tube by gentle sucking, and then remove the mouth attachment from the mouth. The tissue will remain in position. Otherwise, tissues are frequently either expelled or swallowed as the dissector suddenly remembers to breathe. To place the tissue on the collagen bed, it should be sufficient to touch down the capillary tip onto the collagen bed. Otherwise, place mouth attachment back in mouth, and expel very gently. Do not blow hard—the tissue will fragment. Sufficient medium should be transferred with the explant(s), so that they do not dry out. However, the medium should stay on the collagen bed: if too much medium is added and flows off the bed, the second layer of collagen will also flow off the bed, and explants cannot be appropriately positioned.

13. Keep the gelling collagen on ice, so it does not set too quickly. When adding the top layer of collagen, explants often float to the top of the drop. This will be minimized if explants are left in a tiny amount of medium and are not allowed to dry out. Nevertheless, even when explants do float to the top of the drop, they can and must be pushed back down onto the collagen and repositioned. With practice,this can be accomplished with the tungsten needle without tearing even fragile tissue. It is important that the tissue comes to lie on the bottom collagen bed, rather than within the top cushion. Also note that the collagen cushion and top collagen will become alkaline (pink) while setting. This has never been found to affect experiments, although it is possible that some tissues may be sensitive to the alkalinization. The top layer of collagen should overlap the bottom collagen bed precisely (see Fig. 1).

14. When establishing collagen gel cultures, first practice manipulating only a single explant within a gel. With practice, many (up to about 10) explants (approx 100-200 | m2) can be positioned within a single bed. Explants can be positioned in experimental configurations relative to each other as the collagen sets. For instance, explants can be intertwined if contact is required between them or can be positioned apart. Manipulation of the explant(s) is facilitated if it lies in the center of the collagen bed.

15. The type of medium used varies according to the explants being cultured. Neural tissue survives well in Opti-MEM (Gibco-BRL), especially if supplemented with small quantities of sera. Serum screening should be performed, since certain batches adversely affect embryonic tissue in an apparently species-dependent fashion. Medium should be added to the top of the collagen, rather than to one side, to prevent the two layers from separating.

16. Neural explants survive well within collagen cultures for at least 5 d. Often, necrosis is observed early on in culture around the cut edges of the explanted tissue.

17. The collagen gels are composed of fibers that are not so dense that they preclude the diffusion of small growth and differentiation factors. Nevertheless, the isolation of collagen is sufficiently crude that the cultures may be contaminated with extracellular matrix components. Although there are no documented examples, it remains possible that these may affect the properties or presentation of growth factors within the cultures.

18. The resuspension volume can vary considerably. In different experiments, the number of cells per hanging drop (20 |L) has varied between 500 and 20,000 (4,9).

19. To freeze in OCT, place a small amount of OCT on a chuck and place on dry ice. As soon as the OCT begins to freeze, remove the chuck from the dry ice, and add a small drop of OCT. Quickly place the collagen in the center of this drop, and transfer back to dry ice to freeze. Often it is unecessary to push the collagen into the OCT—sufficient OCT flows over it by capillary action.

20. If the tissue explants are very small (around 50- 200 |im2), the entire explant will be collected in only a few sections. Appropriate care must therefore be taken to ensure every section is collected.

21. The variables that must be considered are the same as those considered for standard in situ hybridization of embryos—temperature, probe concentration, and washing stringency must all be optimized. For some probes, better results are obtained if the explants are removed from either one-half or all of the collagen after fixation and processed in small vials.

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