LacZ Expression by PGalactosidase Immunostaining

2. Paraformaldehyde (4%) in Ca2+ and Mg2+-free PBS (see Subheading 2.4, items 1 and 2 for preparation).

4. OTC embedding fluid (Tissue Tek, Elkhart, IN, 4853).

5. Liquid nitrogen.

6. Avertin (2.5%; see Subheading 2.4., item 13 for preparation).

7. Sodium nitrate (Sigma S5506): Make up as 0.5% solution in 0.5X PBS.

8. Vibratome (Campden Instruments, Loughborogh, UK).

9. Sodium azide (Sigma S2002).

10. Normal horse serum (Trace, Sydney, NSW, Australia, 150400100v).

11. Triton X-100.

12. Escherichia coli P-galactosidase antibody rabbit IgG (1° antibody; Cappel 55976).

13. Biotinylated goat antirabbit IgG (2° antibody: Vectastain Elite ABC kit; Immunodiagnostic, Bonsheim, Germany, PK6101).

14. Texas red conjugated donkey antirabbit IgG (2° antibody; Jackson Immuno Research, West Grove, PA).

15. Streptavidin-peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories; Immunodiagnostic, Burlingame, CA, PK6101).

16. Diaminobenzedine (DAB: Dako, Carpinteria, CA, S3000). Store at -20°C.

17. Hydrogen peroxide (H2O2)

18. Streptavidin-FITC complex.

19. Nuclear fast red (0.1%; see Subheading 2.4., item 12).

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