What is in situ hybridization
Radioactive ISH
Fluorescent ISH
Colorimetric ISH
ISH: three variables
The sample
The probe
Optimizing ISH Detection
ISH controls
Data Analysis
1. Session 2:Session 2:
In situ analysesIn situ analyses
mRNA and Protein expressionmRNA and Protein expression
in archival clinical specimensin archival clinical specimens
Fishing for genes in cells and tissues
Abizar Lakdawalla
Abizarl@biogenex.com
925-543-1408
2. ContentsContents
1. What is in situ hybridization
2. Radioactive ISH
3. Fluorescent ISH
4. Colorimetric ISH
5. ISH: three variables
6. The sample
7. The probe
8. Optimizing ISH Detection
9. ISH controls
10.Data Analysis
3. In situIn situ HybridizationHybridization
In situ = Inside (cell/tissue)
Hybridization = Specific Binding of a Probe
Detection = Visible Reaction
To identify a specific genes (DNA or RNA) in intact
cells, tissues or even whole animals.
5. In situ HybridizationIn situ Hybridization
ISH -
Detection of specific nucleic acid
sequences (signatures) within cells
and tissues by “hybridizing” a
complementary probe.
Uses -
Finding pathogens, a specific gene, a
mutant gene, cells that have certain
genes switched on.
Part I Optimization of in situ detection methods
6. In situ HybridizationIn situ Hybridization
Radioactive in situ hybridization
(simple but time consuming and hazardous)
Fluorescent in situ hybridization
(simple, quick but short-lived results)
Colorimetric in situ hybridization
(simple, quick and long-lived results)
7. Radioactive ISHRadioactive ISH
Protocol SummaryProtocol Summary
Dewax Slides
Permeabilize, target retrieve & Post-fix
Denature and Hybridize radiolabeled-Probe
Post-Hybridization Washes
Counterstain
Photographic emulsion
Expose for days to weeks
Develop
Read
18. C-ISH v/s FISHC-ISH v/s FISH
CISH
Detect mRNA and DNA
DNA located in the
nucleus
RNA located in the
cytoplasm
Colored end-point
Can be archived
Greater comfort level for
pathologists
Cannot detect more than
2 genes
RNA degrades easily
FISH
Mostly DNA detection
DNA located in the
nucleus
Fluorescent end-point
Cannot be archived
Hard to read morphology
Can detect multiple genes
simultaneously
DNA does not degrade
19. Just
like
IHC
C-ISH Protocol SummaryC-ISH Protocol Summary
Dewax Slides
Permeabilize & Post-fix
Denature and Hybridize Probe
Post-Hybridization Washes
Block
Anti-Probe Antibody
Secondary Antibody
Streptavidin-Enzyme
Substrate/Chromogen
Counterstain
Mounting Medium
24. ISH variablesISH variables
The tissue
Frozen or fixed
Pretreatment or not
The probe
Sequence selection
RNA or DNA
single or double stranded
type of label
The Detection
Fluorescent, Colorimetric
25. In situ assays:In situ assays:
Three main variablesThree main variables
I.
SAMPLE
III.
DETECTION
II.
MOLECULAR
PROBE
Most
important
Medium
importance
Least
important
26. 10-40um
Sample Type:Sample Type:
MonolayersMonolayers
1. Layer of cells grown on a solid substrate
2. Suspension cells that have been
concentrated onto a solid substrate
(cytospins)
3. Smears of a fluid prep (blood smears)
Weak Staining
if membrane
not removed
1. preparing the tissue
27. 5um
Sample Type:Sample Type:
Tissue SectionsTissue Sections
Blocks of excised tissues that are
fixed and embedded in a wax or
resin or frozen and then cut into
thin sections.
Sometimes tissues imprints are
also used.
Weak Staining
as only part of
cell available
1. preparing the tissue
28. Effects of FixationEffects of Fixation
Fixation Embedding IHC
Reactivity
ISH
Reactivity
Frozen Sucrose/OCT ++++ ++++
Chemical
fixation
Paraffin ++ +++
Chemical
fixation
Methacrylate +/- +++
Chemical
fixation
Epoxy - +
1. preparing the tissue
29. ISH Sample prepISH Sample prep
Sample preparation is more forgiving
than in IHC (most standard fixatives
work well)
Adequate permeabilization is
important (pretreatment with
proteases or Target Retrieval)
Contamination with RNase’s should
be avoided (some solutions need to be
RNase free)
30. EZ-DeWax
ReagentV/S
Dewaxing and rehydrationDewaxing and rehydration
in one step!in one step!
1. Xylene
2. Xylene
3. Xylene
4. 100% EtOH
5. 100% EtOH
6. 90% EtOH
7. 80% EtOH
8. 70% EtOH
(5 min each)
1. preparing the tissue
31. Optimizing ISH:Optimizing ISH:
Sample pretreatmentSample pretreatment
Target Retrieval and/or protease
digestion
removal of proteins that coat nucleic
acids
increases access into the cell
32. Nucleic Acid Retrieval (NAR-1)Nucleic Acid Retrieval (NAR-1)
Combines deparaffinization and
replaces proteinase K digestion
Enhances penetration of probes to
target molecules
The NAR-1 protocol is
recommended for use with tissues
fixed with formalin only and DNA
targets.
33. Optimizing ISH:Optimizing ISH:
Sample pretreatmentSample pretreatment
Indirect evaluation of protease digestion
(Tissue stained with propidium iodide).
If clear distinct fluorescent nuclei without
much cytoplasmic staining then the digestion
is appropriate.
If cytoplasm is fluorescent, then the tissue is
underdigested.
If nuclear margins are indistinct, then tissue
is overdigested.
In situ
detection:
ISH
34. Optimizing ISH:Optimizing ISH:
Sample pretreatmentSample pretreatment
Direct evaluation of protease digestion
(By titration).
Concentration of Protease
0x 0.5x 1x 5x
Staining 11 22 4 33
Morphology 44 44 3 11
In situ
detection:
ISH
35. ISH: The ProbeISH: The Probe
Types of Probes
Types of Tags or
Labels on the probe
Location of the Labels
Labeling methods
In situ
detection:
ISH
36. ISH: Types of ProbesISH: Types of Probes
Double Stranded (weaker signal)
Random primed probes
Nick translated probes
Single Stranded (stronger signal)
Transcribed RNA probes
M13 probes
Asymetrical PCR probes
Oligonucleotides
In situ
detection:
ISH
37. ISH: Types of LabelsISH: Types of Labels
Direct Labels
Fluorescent dyes attached to the probe
Enzymes attached to the probe
Indirect Labels (detected by antibodies)
Biotin
Fluorescein
Digoxigenin
In situ
detection:
ISH
38. ISH: Preparing ProbesISH: Preparing Probes
Enzymatic methods
Nick translation by DNA polymerase
Random priming by viral DNA polymerase
3’-end Labeling and Oligo tailing with TdT
End labeling with DNA Ligase
PCR extension
Transcription by RNA polymerase
Chemical methods
Oligo synthesis with labeled amidites
Post-synthesis chemical ligation
Photoactivated label addition
40. Optimizing ISH:Optimizing ISH:
Probe concentrationProbe concentration
Check Labeling
Efficiency
Titer probe by
performing ISH on
control tissue (range
varies from
picomoles to high
nanomoles).
41. Nucleic acid target
located inside the cells
probe
Fluorescein
tags
Hybridization of ProbeHybridization of Probe
Nucleic acids (DNA and RNA) are made up of four
building blocks (nucleotides = A, T, G, C).
A piece of DNA (probe) which has the complementary
sequence to a target (A replaced by T, T by A, G by C,
and C by G) will bind to the target quite strongly.
The probe is added to the slide, warmed (to separate the
target DNA strands) and cooled so the probe can bind to
it’s target.
42. Optimizing ISH:Optimizing ISH:
Hybridization ConditionsHybridization Conditions
Maximal hybridization efficiency occurs at
25C less than the denaturation temperature of
DNA (Tm).
Tm = 81.5 +0.41(GC) - 0.72 (% formamide) for
Na+ greater than 0.4M
For Na+ less than 0.2M add (16.6 log M (Na+))
to the above equation.
For Oligo’s, 25C with a 25% formamide buffer
is optimum. For probes >100nt, 37-45C with a
50% formamide buffer is optimum.
43. Hybridization of ProbeHybridization of Probe
Rule – of – thumb conditions
For small probes (oligos)
25-35C for 1-4 hours hybridization
Wash in 0.5x PBS or SSC at 35C
For large probes
45-65C for 6-16 hours
Wash in 0.2x PBS or SSC at 65C
44. Optimizing ISH:Optimizing ISH:
Post-hybridization washesPost-hybridization washes
25C 35C 45C 55C 65C
Staining 4 4 4 3 0
Background 4 4 1 0 0
1x PBS with 0.1% Tween-20 was used as the wash buffer.
Probe was a cocktail of 5 oligo’s of 24-28 mer.
46. Optimizing ISH: ControlsOptimizing ISH: Controls
Negative Controls
1. No Probe
2. No Probe, Anti-probe Antibody
3. Omit Pr, Anti-Pr, Secondary Antibody
4. Omit Pr, Anti-Pr, Sec.Ab., Streptavidin-
Enzyme
5. Replace Anti-Sense probe with Sense
1 2 3 4
No ProbeProbe No Antibody No Sec. Antibody No Enzyme
47. Optimizing ISH: ControlsOptimizing ISH: Controls
Negative Controls
Omit Probe
Omit Anti-probe Antibody
Omit Secondary Antibody
Omit Streptavidin-Conjugate
Replace Anti-Sense probe with Sense
Positive Controls
ISH with a housekeeping gene
ISH with cells known to express the sequence
52. Data Analysis of ISH in TMA’sData Analysis of ISH in TMA’s
Based on a four slide experiment
Slide 1: stained with Neg Ctrl probe
(random probe or Sense controls)
Slide 2, 3, 4: stained with three
conditions of test probe
Slide 5: stained for GAPDH
Slide 6: stained for B-Actin
Obtain cell-compartment-specific
staining intensities (nucleus, cytoplasm,
membrane)
Subtract background staining intensities
(from Neg Ctrl)
Obtain slope values for test probe.
Divide with housekeeping gene values
for each element (intensity of test
gene/intensity of HK gene)
Correlate with cellular phenotype
Correlate with clinical information
