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
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
PCR
- RNA : in vitro transcription , RT- PCR
- oligonucleotides : endlabeling or tailing
translation ,
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 , BiotinddUTP ,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
Treatments to prevent background staining
- endogenous enzyme inactivation
- RNase-treatment
b) Permeabilization
- diluted acids
- detergent/alcohol
- proteases
a)
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 alpherpheto-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