1. All sera (fetal bovine, calf, horse) are suitable for use in freezing media.

2. Chloroquine increases the retroviral titer (8).

3. Paraformaldehyde will dissolve in approx 20 min with heating and stirring.

4. K3Fe(CN)6 and K4Fe(CN)6 are commonly used at concentrations between 5 and 20 mM. The higher concentrations may result in increased staining intensity.

5. This pH range is optimal for detection of the bacterial lacZ activity. The use of higher pHs should be avoided so as to minimize the endogenous mammalian P-galactosidase activity.

6. The chloral hydrate should be made fresh on the day of surgery; stored chloral hydrate can be used for anesthetizing rats or mice prior to perfusion. Supplementary doses of chloral hydrate should be avoided because death of the experimental animal may occur. Before giving any additional chloral hydrate allow additional time for the chloral hydrate to achieve its effect. As an alternative to using chloral hydrate, equithesin can be used.

7. A heat-absorbing lens should be attached to the end of each fiber optic light guide to reduce the desiccation of exposed tissue, and in particular the uterine swellings, during surgery.

8. Different instruments should be used for surgical and perfusion procedures; traces of fixative may be deleterious to living tissue.

9. The experimental animals must be kept warm post surgery so that they do not succumb to the temperature lowering effects of anesthesia. Cover half of the cage with aluminum foil to reduce the temperature generated by the infrared heat source. Therefore, upon awakening from anesthesia the experimental animal can choose to remain under the heat source or to go under the aluminum foil heat shield. Typically the anesthetized pregnant dams awaken within 30-45 min.

10. As an alternative to sectioning with a Vibratome, the tissue can be sectioned on a cryostat and mounted on glass slides.

11. The use of Superfrost slides eliminates the need to sub the slides.

12. A retrovirus with a nuclear localization signal can be used instead so that lacZ expression is restricted to the nucleus (18). This allows the cytoplasmic expression of other antigens to be detected more easily.

13. Choose the acetate sheets carefully. Baking will ruin many kinds of acetate and make then useless for embedding. Avoid acetate sheets that become opaque when baked or that stick permanently to the polymerized Araldite.

14. Growing cells to confluence results in a number of unwanted changes in the producer cells, among them aneuploidy and increased frequency of DNA recombination events.

15. The volume is decreased in order to increase the relative retroviral titer.

16. The half-life of retroviral particles at 37°C is approx 4 h.

17. Care should be taken during media changes as the Bosc23 cells are loosely adherent.

18. Decreasing the temperature to 32°C during collection of the supernatant increases the retroviral titer 5- to 15-fold (19).

19. After 72 h from the start of transfection the titer of infectious retrovirus greatly drops.

20. The efficiency of transfection should approach 50%.

21. The length of fixation is critical. LacZ activity drops dramatically if the cells are fixed for long periods.

22. The diluted X-gal in staining buffer can be reused several times.

23. The cells should be subconfluent before infecting with virus. Retroviruses only infect replicating cells.

24. Use a new pipet to aspirate each well so as not to transfer any retrovirus into neighboring wells.

25. If the titer of wild-type retrovirus in the test supernatant is low, very few 3T3 cells will become infected. Continued growth of the cultures allows time for the wild-type retrovirus to infect other 3T3 cells in the dish increasing the likelihood of detecting its presence. Low levels of polybrene in the media help the viral infection of neighboring cells.

26. If the test supernatant contained wild-type retrovirus, then the 3T3 cultures now produce the wild-type retrovirus themselves after several days. Thus, the supernatant from the 3T3 cells is capable of infecting a naive set of 3T3 cells.

27. For testing the presence of a wild-type retrovirus expressing a neomycin resistance gene, after 2-3 d, split the second set of 3T3 cells at a 1:20 dilution into GM with 1 mg/mL G418. Change the media after 3 d and count colonies after 10 d. If the retroviral producer cells express only replication defective virus, there should be no colonies.

28. Retrovirus can also be injected into the amniotic cavity to label structures that can not be seen, but are in direct contact with the amniotic fluid, such as skin.

29. If a pneumatic picopump is not available, then alternatively, a Hamilton syringe can be loaded in the usual way, and used to deliver retrovirus.

30. The pipet tip can be backfilled with the colored water or viral supernatant. Alternatively, 5-10 |L of the viral supernatant can be deposited on a small plastic boat or piece of parafilm, and then with extreme care, the pipet tip gently placed in the center of the droplet at an angle in order to load the pipet by vacuum.

31. Work as quickly as possible once the animal has been anesthetized because the anesthesia can deleteriously lower body temperature. The investigator should strive to finish the surgery within 1.5 h.

32. The surgery platform serves two principal purposes. First, it aids in restricting the pregnant dam in a position in which her abdomen is accessible to surgery. Second, it raises the anesthetized dam above the base or tray, where the irrigation fluids will drain. This helps to keep the dam from laying in a pool of fluid, which would adversely lower the body temperature and curtail recovery.

33. While shaving the fur off the ventral surface of the abdomen, carefully shave around the nipples so that they are not injured. If the nipples are malfunctioning because of surgical trauma, the neonatal animals cannot suckle.

34. Be sure the alcohol has dried before incising the abdomen because alcohol can be irritating to an open wound.

35. If the uterine membranes dry out, they become opaque and resistant to penetration by the pipet tip. If the area of the abdominal incision dries out, a clean closure of the tissue is difficult to achieve, and subsequently the pregnant dam is more at risk of infection.

36. The number of uterine swellings present varies from animal to animal. Mice usually have between 7 and 14, whereas rats usually have between 11 and 17. Be careful not to mistake the bladder for a uterine swelling or to maneuver it any more than necessary.

37. A uterine swelling containing a dead fetus or one that is being resorbed is usually gray rather than pink, and considerably smaller than the healthy looking uterine swellings. The vascularization of the healthy uterine swellings are more prominent. Commonly one or more of the uterine swellings may contain a dead or resorbing fetus.

38. The end of the fiber optic light source can emit heat and surprisingly quickly dry out the tissues in its proximity if the tissues are not repeatedly moistened.

39. When not in use, immerse the vial of retrovirus in the ice bucket. The virus will only remain viable for a couple of hours if maintained on ice.

40. While tilting or raising the embryo within the uterine swelling, be careful not to puncture the uterine membranes or to apply excessive pressure to the embryo.

41. Do not apply excess force to enable the pipet tip to penetrate the uterine wall. If the tip will not go through easily, greater pressure will just cause the pipet tip to snap off. Instead, try to puncture the uterine wall in a slightly different place, at a slightly different angle, or with a faster motion.

42. The following procedure can be modified to perfuse injected embryos by first anesthetizing the pregnant dam and making a midline abdominal incision, similar to that done in advance of the laparoscopic surgery for retroviral injections. Expose the uterine horns. Incise the uterine membranes and remove the injected embryo by disconnecting it from placental circulation. The injected embryos can be perfused as described below or fixed by immersion in the fixative. Remove an embryo and fix it before recovering and fixing additional embryos.

43. If desired, the descending aorta can be clamped.

44. If perfusing embryos, then use a small syringe needle to deliver the fixative to the left ventricle. Butterfly needles of various gages connected to the tubing of the peristaltic pump work well to perfuse embryos as young as E12 mice and E13 rats.

45. The peristaltic pump can be turned on in advance of snipping the right atrium so that the fixative is flowing at the time the cannula is inserted into the left ventricle.

46. If the brain is left in the fixative for more than 1-1.5 h, the lacZ may start to decay thereby reducing the intensity of the histochemical reaction product.

47. Alternatively, a secondary antibody conjugated to an enzyme such as horseradish peroxidase or alkaline phosphatase followed by incubation in the appropriate substrate can be substituted.

48. Multiple sections can be placed in the same well.

49. Check to make sure that the osmium tetroxide is fully dissolved. Osmium crystals must be left overnight in distilled H2O to dissolve, and then mixed the following morning with 0.2 M phosphate buffer to make a working solution of 1% OsO4. Remember to wear gloves and work in the hood when handling osmium tetroxide.

50. Make sure the sections are flat and not overlapping. The OsO4 solution will turn the tissue sections a dark color and make them somewhat brittle.

51. All experimental procedures must comply with legal restriction of the appropriate country (see also Notes in Chapter 4).

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