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Slides preparation

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). probe. a) Choice of the probe - ds : DN0A , cDNA - ss : RNA , oligonucleotide ss-DNA

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Slides preparation

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  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

  21. The DIG system

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

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

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

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

  26. Quantitation by image scan

  27. Color transformation for digital image analysis

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

  29. The stages of Image analysis for ISH Result

  30. Immunohisto Chemistry of GRB2

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