slides preparation
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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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)
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probe
a) Choice of the probe -ds : DN0A , cDNA
-ss : RNA , oligonucleotide ss-DNAb) 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
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Oligonucleotide 3’ –end labeling with DIG-ddutp , Biotin-ddUTP ,or Fluorescein-ddUTP
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PCR DIG labeling reaction for highly labeled probes containing unique sequences
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Oligonucleotide 5’ –end labeling with DIG-ddutp , Biotin-ddUTP ,or Fluorescein-ddUTP
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DIG Tailing System
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Non –Radioactive Random Primed Labeling
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A PCR Strategy for Rapid Generation of Template DNA for Synthesis of Labeled RNA probes
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Consensus Promoter Sequences . The +1base is the first base incorporated into RNA during transcription .The underline indicates the
minimum sequence required for efficient transcription
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RNA labeling by in vitro transcription of DNA with DIG , Biotin or Fluorescein RNA Labeling Mix
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Estimating the yield in a spot test with a DIG-labeled control
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Specimen pretreatment
a) Treatments to prevent background staining
- endogenous enzyme inactivation
- RNase-treatment
b) Permeabilization
- diluted acids
- detergent/alcohol
- proteases
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Determination of hybridization conditions, e.g,.
- determination of hybridization temperature , pH ,use of formamide , salt concentration
- composition of hybridzation solution - Probe concentration
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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
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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-
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post-hybridization steps
- treatment with single strand specific nuclease(optional)
- strigency washes
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Detection
- blocking step
- antibody incubation
- colorimetric substrate for fluorescence microscopy
- counterstaining
- mounting
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NBT / BCIP Color Substrates and Reaction Products
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In situ hybridization of H19 rat probe to Mouse embryo using Anti sense probe
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In situ hybridization of H19 rat probe to Mouse embryo using sense probe
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The DIG system
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Typical ISH for H-19 gene product in TCC Sample
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Hepator cellular carcinoma stained with H19 and alpher-pheto-protien.
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Tcc at different cancer grades with increasing expression of H19 gene
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Typical ISH for H-19 gene product in TCC Sample
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Quantitation by image scan
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Color transformation for digital image analysis
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Standard carne with increasing probe concentration at the same slide
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The stages of Image analysis for ISH Result
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Immunohisto Chemistry of GRB2