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.

4. DNADNA
RNARNA
ProteinProtein
IHC
IHCISH

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

8. In situ hybridizationIn situ hybridization
Target Gene
(DNA)
Probe
P33
, S35
-
labeled

9. Radioactive inRadioactive in
situsitu
Hybridization inHybridization in
Normal andNormal and
Tumor cellsTumor cells

10. RadioactiveRadioactive
ISHISH

11. Fluorescent ISHFluorescent ISH
Protocol SummaryProtocol Summary
Dewax Slides
Permeabilize, target retrieve & Post-fix
Denature and Hybridize fluorescent labeled-
Probe
Post-Hybridization Washes
Counterstain
Fluorescence microscopy

12. In situ hybridizationIn situ hybridization
Target Gene
(DNA)
Probe
Fluor-
labeled

13. FISHFISH

14. Fluorescent ISHFluorescent ISH
(usually for DNA targets)(usually for DNA targets)

15. N-myc amplificationN-myc amplification
in neuroblastomain neuroblastoma

16. N-myc and EGFRN-myc and EGFR
in TMA nucleiin TMA nuclei

17. HER-2/neu amplificationHER-2/neu amplification
cen17
HER-2

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

20. Primary Antibody
Secondary Antibody
Streptavidin-
Conjugate
Colorimetric In situ hybridizationColorimetric In situ hybridization
Chromogen
Target Gene
(mRNA)
Probe

21. HPV16/18HPV16/18

22. HPV16/18HPV16/18

23. C-ISHC-ISH
ProbesProbes
HPV 6/11HPV 6/11
EBEREBER
KAPPAKAPPA
LAMBDALAMBDA
CYCLINCYCLIN
D1D1
RBRB ALU IIALU II

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

39. ISH SensitivityISH Sensitivity
Concentration of Probe
Length of Probe
Number of Labels on the Probe
Amount of target (DNA or mRNA)

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.

45. Which chromogen?Which chromogen?
Chromogen Enzyme Permanent Fluorescent Sensitivity Counter-
stain
Contrast
AEC Per. - - ++ ++++
BCIP/
NBT
Phos. + - ++++ ++
DAB Per. + - ++++ ++++
Fast
Red
Phos. - + +++ ++++
TMB Per. + - +++ ++

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

48. Optimizing ISH:Optimizing ISH:
Negative controlNegative control
ISH with
kappa probe
ISH with
kappa probe
after treating
sample with
RNAse

49. In Situ Hybridization
Tissue & probe
preparation
Hybridization
Signal
visualization
Tissues:
- Fixation
- Protease
digestion
- Denaturation
-Oligonucleotides
-ssDNA
-dsDNA
-ssRNA
Radioactive label
F-label
Dig-label
Biotin-label
Probes:
Prehybridization
Hybridizaiton
-time
-temperature
post-wash
-temperature
-salt concent.
-time
Radioactive probe:
-Autoradiography
Fluorescent-labeled
probe: FISH
-directly viewed
under F-microscopy
Dig, Fluor or biotin-
labeled-probe: CISH
-indirectly detected by
IHC

50. Primary Antibody
Poly-HRP conjugated
Secondary Antibody
Chromogen
Super sensitive non-biotin HRP
detection system

51. AutomationAutomation
Tissues/
Tissue arrays
Pretreatment
Reagents
Antibodies/
Probes
Detection
Reagents
Automation
Slide labelSlide label
printerprinter
(i500)(i500)
An RetrievalAn Retrieval
(i1000)(i1000)
Slide StainerSlide Stainer
(i6000)(i6000)
Slide imagerSlide imager
(iVision)(iVision)
Barcoded

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