Slides preparation

1. Slides preparation - e.g., gelatin or poly – lysine treatment of slides • siliconization of coverslips • by precipitation ( e.g., ethanol) • by cross-linkage (e.g., formaldehyde)

2. probe a) Choice of the probe - ds : DN0A , cDNA - ss : RNA , oligonucleotide ss-DNA b) Preparation of the probe - DNA : fragment isolation (optional) - cDNA : cloning - RNA : cloning in transcription vectors - oligonucleotides c) Labeling of the probe - ds DNA : random primed DNA labeling nick translation , PCR - RNA : in vitro transcription , RT- PCR - oligonucleotides : endlabeling or tailing

3. Oligonucleotide 3’ –end labeling with DIG-ddutp , Biotin-ddUTP ,or Fluorescein-ddUTP

4. PCR DIG labeling reaction for highly labeled probes containing unique sequences

5. Oligonucleotide 5’ –end labeling with DIG-ddutp , Biotin-ddUTP ,or Fluorescein-ddUTP

6. DIG Tailing System

7. Non –Radioactive Random Primed Labeling

8. A PCR Strategy for Rapid Generation of Template DNA for Synthesis of Labeled RNA probes

9. Consensus Promoter Sequences . The +1base is the first base incorporated into RNA during transcription .The underline indicates the minimum sequence required for efficient transcription

10. RNA labeling by in vitro transcription of DNA with DIG , Biotin or Fluorescein RNA Labeling Mix

11. Estimating the yield in a spot test with a DIG-labeled control

12. Specimen pretreatment • Treatments to prevent background staining - endogenous enzyme inactivation - RNase-treatment • Permeabilization - diluted acids - detergent/alcohol - proteases

13. Determination of hybridization conditions, e.g., • determination of hybridization temperature , pH ,use of formamide , salt concentration • composition of hybridzation solution • Probe concentration

14. Prehybridization Incubation of specimen with a pre-hybridization solution (= hybridization solution minus probe) is performed at the same temperature as hybridization • denaturation of probe and target • pH or heat • simultaneous or separate denaturation of probe and target ( if double stranded

15. Hybridization Components of the solution are mainly : • Denhardt’s Mix ( Ficoll, BSA, PVP) • heterologous nucleic (e.g., herring sperm DNA / tRNA / competitor) • sodium phosphate, EDTA, SDS, salt • formamide • dextran sulfate

16. post-hybridization steps • treatment with single strand specific nuclease(optional) • strigency washes

17. Detection - blocking step - antibody incubation - colorimetric substrate for fluorescence microscopy - counterstaining - mounting

18. NBT / BCIP Color Substrates and Reaction Products

19. In situ hybridization of H19 rat probe to Mouse embryo using Anti sense probe

20. In situ hybridization of H19 rat probe to Mouse embryo using sense probe

22. The DIG system

23. Typical ISH for H-19 gene product in TCC Sample

24. Hepator cellular carcinoma stained with H19 and alpher-pheto-protien.

25. Tcc at different cancer grades with increasing expression of H19 gene

26. Typical ISH for H-19 gene product in TCC Sample

27. Quantitation by image scan

28. Color transformation for digital image analysis

29. Standard carne with increasing probe concentration at the same slide

30. The stages of Image analysis for ISH Result

31. Immunohisto Chemistry of GRB2