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-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

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

PCR DIG labeling reaction for highly labeled probes containing unique sequences

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

DIG Tailing System

Non –Radioactive Random Primed Labeling

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

Consensus Promoter Sequences . The +1base is the first base incorporated into RNA during transcription .The underline indicates the

minimum sequence required for efficient transcription

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

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

Specimen pretreatment

a) Treatments to prevent background staining

- endogenous enzyme inactivation

- RNase-treatment

b) Permeabilization

- diluted acids

- detergent/alcohol

- proteases

Determination of hybridization conditions, e.g,.

- determination of hybridization temperature , pH ,use of formamide , salt concentration

- composition of hybridzation solution - Probe concentration

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

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-

post-hybridization steps

- treatment with single strand specific nuclease(optional)

- strigency washes

Detection

- blocking step

- antibody incubation

- colorimetric substrate for fluorescence microscopy

- counterstaining

- mounting

NBT / BCIP Color Substrates and Reaction Products

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

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

The DIG system

Typical ISH for H-19 gene product in TCC Sample

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

Tcc at different cancer grades with increasing expression of H19 gene

Typical ISH for H-19 gene product in TCC Sample

Quantitation by image scan

Color transformation for digital image analysis

Standard carne with increasing probe concentration at the same slide

The stages of Image analysis for ISH Result

Immunohisto Chemistry of GRB2