Notes

1. When crosslinking the DNA to the membrane, UV exposures >5 min will appreciably diminish the signal. As an alternative to UV exposure, baking the membrane at 120°C for 30 min will enable an equally sensitive detection.

2. For detecting high-copy-number transgenics, a positive result can be seen in as little as 1-10 ng of genomic DNA. For low-copy-number transgenics, a positive result can be seen after 15 min for 5-19 |g genomic DNA. A distinct result can be seen after 1.5-2 h.

3. Chemiluminescent detection produces a permanent record. The colorimetric reaction is equally sensitive, but the color reaction will fade with time.

4. The sensitivity of the detection can be improved by changing the wash stringency and probe concentration. The conditions shown here will minimize backgound in relation to signal. However, a clear background may not be possible for low-copy-number transgenic samples.

5. Enzyme activity in embryos from preimplantation up to about 11.5 d p.c. can be directly analyzed in whole mounts. However, for embryos older than 11.5 d penetration of the substrate in whole embryos becomes limiting and this poses a problem for accessibility of staining reagent to internal tissues, especially after a substantial amount of insoluble reaction product has been deposited in more superficial lacZ-expressing tissues. Dissection of older embryos (>11.5 d) into smaller fragments facilitates penetration of the substrate and greatly improves staining of deeper tissues.

6. In order to reduce the background associated with endogenous P-galactosidase activity which is normally found in bone, kidney, and brain, the histochemical reaction should be carried out at pH 7.4, which is optimal for bacterial P-galactosidase in contrast to the mammalian enzyme, which is most active at a more acidic pH value of 4.0.

7. Prolonged exposure of embryonic tissues to the fixative diminishes P-galactosi-dase activity, and enzyme activity becomes undetectable after more than 60 min in paraformaldehyde.

8. If the fixation of the embryo fragment/specimen is insufficient, leakage of the reaction product stains the reaction mixture blue. It is advisable not to leave stained samples in alcohols, and solvents longer than necessary, since this may leach out some of the reaction product. When sections are viewed by dark-fleld microscopy, the lacZ stain appears pink and contrasts with surrounding tissues.

9. In order to enhance the discrimination between expressing, nonexpressing cells, and cells expressing endogenous P-galactosidase-like activity, it is useful to target the lacZ product to a particular cellular compartment, such as the nucleus. The nuclear localization signal (nls) from the early region of the simian virus (SV40) genome has been used to provide effective nuclear localization of P-galactosidase (nlsLacZ; 17,18).

10. For more detailed histological studies, small parts of the organ are cut by scalpel blade, dehydrate in alcohol, and embedded either in paraffin wax or glycol meth-acrylate. Wax sections are counterstained in 0.1% nuclear fast red. Methacrylate sections are counterstained in 0.1% cresyl violet in 1% acetic acid (pH 3.3) for 50 min at 40°C.

11. When infiltrating with polyester wax, the specimens may float, which is normal, but they will sink as they are infiltrated with wax. If after the first 15 min of incubation in wax the specimens have not sunk, it is still advisable to go to the next wax change.

12. It is important that the embryos are not overfixed with paraformaldehyde for X-gal staining prior to the immunohistochemistry, because it may result in some loss of antigenicity.

13. In each of these procedures, there are a lot of wash steps that require the transfer of embryos. If the dish containing the embryos is placed on a black background, it is easier to see the embryos during transfer.

Was this article helpful?

0 0

Post a comment