GREEN marks the position of the protein fibronectin in this frog embryo Section HOW DO WE

1. GREEN marks the position of the protein fibronectin in this frog embryo Section HOW DO WE DO THIS? RED marks nuclei

2. Making Polyclonals serum + or - purif.

3. MCB 6.2(‘0601’) Monoclonals

4. PAGE PolyAcrylimide Gel Elecrophoresis MCB 3 Western or Immunoblot MCB 3.5 “3EIMMBLOT”

5. Immunocytochemistry Most common enzyme conjugates: Alkaline phosphatase Horseradish Peroxidase

6. Fluorescent microscopy FITC secondary

7. Anti-fibronectin Then FITC Fluorescence, rather than a converted substrate, as secondary to mark protein’s presence RED, PI, nuclear counterstain

8. Principle of Confocal microscopy

9. Confocal – What it offers Fluorescence microscopy Regular Confocal

10. ActinGurken

11. Hunchback Kruppel

12. Immunogold SO: Immunogold Immunofluorescence Immunocytochemistry

17. Tracking specific macromolecules For protein: antibody-antigen For nucleic acid: n.a. complementarity

18. MCB 7.2 PCR Start here week2/3

23. In situ hybridization with 35S RNA probes 1 2 3 4

24. In situ hybridization using radioactive probe -expose photographic emulsion

27. In situ –Shh

28. FGF8 in situ

29. FISH

30. Northern Northern or SLOT-BLOT

31. Northern - or dot/slot blot Developmental Northern or SLOT-BLOT Microarray Tiling Microarray RNA seq. Single cell RNA seq. RIBOSOME PROFILING- on way to proteome

32. MICROARRAY ANALYSIS

33. Array analysis: see animation from Griffiths

34. Figure 4.16(1) Microarray Analysis of Those Genes Whose Expression in the Early Xenopus Embryo Is Caused by the Activin-Like Protein Nodal-Related 1 (Xnr1)

35. Figure 4.16(2) Microarray Analysis of Those Genes Whose Expression in the Early Xenopus Embryo Is Caused by the Activin-Like Protein Nodal-Related 1 (Xnr1)

36. Figure 4.15(1) Microarray Technique

37. Figure 4.15(2) Microarray Technique

38. Northern - or dot/slot blot Developmental Northern or SLOT-BLOT Microarray Tiling Microarray RNA seq. Single cell RNA seq. RIBOSOME PROFILING- on way to proteome

39. MCB 5.1 Reporter Constructs

40. Myf-5 Driven Beta-gal X-gal

41. Acrosin-GFP

42. GFP spindles http://www.duke.edu/web/microlabs/endow/moviepage.html

44. Other markers of cells

45. Flourecent dye injection into cells

47. Antibody, FITC to P granules Hoescht-Dye

48. A dye that fluoresces when it binds Ca++ Time series