Franceschi Microscopy & Imaging Center
Washington State University
2/17/16
1
In situ Hybridization
Paraffin: Use slides with 1 or 2 sections close together (this reduces the amount of solutions
needed).
Deparaffinization: 2x, 5m - Hemo De
2x, 5m - 100% EtOH
1x, 5m - 50% EtOH, air dry completely, circle
sections with rubber cement to keep solutions on sections and
for sealing coverslip.
Resin: Slides - no special preparations except keep sections close together,
circle
sections with rubber cement to keep solutions on sections and
for sealing coverslip.
Grids - do following steps in microfuge tubes, 50 - 100 ul
Pretreat
Proteinase K (25 mg/ml)
5m, room temperature
(rt)
(0.002g in 1 ml TE buffer)
- protease treatment increases accessibility by digesting protein around target
nucleic acids.
Wash
TE buffer
50 mM TRIS
5 mM EDTA in DEPC water
pH 7.4
- to remove proteinase K
2x, 5m, rt
20x SSC
Deionized Formamide (100%)
3M NaCl
50 ml formamide w/5g mixed bed ion exchange resin
300mM NaCitrate
stir 30m, rt. filter 2x whatman #1 filters
pH 7 (w.10N NaOH) 500 ul aliquots store at -20 C
50x Denhardts Solution
1% PVP
1% Ficoll 400
1% BSA
store at -20C
Dextran Sulfate (50%)
50 mg Dextran Sulfate
65 ul dH2O
Prehybridization
Hybridization solution
+ 1 ul Dextran Sulfate
-prehybridization is to prevent background staining
tRNA
10
10 mg/ml
ssDNA
10 mg/ml
15m, rt-55 C
4. Hybridization (see also notes*)
Hybridization Buffer + probe (1 ng/ul) + 1 ul Dextran sulfate
- temp 30 - 65 C
- time 10 -24 h
- may also preheat probe to 70 C, 5m in water bath to remove secondary structures
Franceschi Microscopy & Imaging Center
Washington State University
5. Washes
Method I
1X SSC
RNase
4X SSC
2X SSC
1X SSC
ddH2O
3x, 5m
20m, rt
5m
5m
5m
jet washes
Method II
4X SSC
2X SSC
1X SSC
0.1X SSC
2/17/16
2
5m, 50 C
5m, rt
5m, rt
5m, rt
-posthybrization washes help remove non-specific hybrids. Probe may bind to sequence which
bears homology but not entirely homologous. Wash with various stringencies, can vary
formamide conc., salt conc., and/or temp.
6. Secondary antibody (if using DIG labeled probe)
Block with TBST-BSA (500 mM NaCl)
30m
Incubate; TBST-BSA : Sheep-anti-DIG
1.5h
1 : 50
Wash; TBST-BSA
10m
TBST
10m
ddH2O
2x, 5m & jet wash
* Notes on hybridization;
hybridization depends on ability of denatured DNA to reanneal with complementary
strands in environment just below their melting point (Tm = temp at which 1/2 DNA is present in
single strand form (denatured)). Stability of DNA directly dependent on GC content, increase
molar ratio of GC pairs = increase melting point (Tm). Renaturation of DNA and Tm influenced
by: temperature, pH, concentration of monovalent cations, presence of organic solvents.
pH: increase pH above 6.5-7.5 to produce more stringent hybridization conditions
Monovalent cations (sodium ions): interact electrostaticly with nucleic acids (i.e.
phosphate groups). Increase salt conc. = decrease electrostatic repulsion between the 2
strands & increase stability of hybridization
Decreased salt concentration affects Tm and renaturation rate.
Formamide: Organic solvent, reduces melting temp of DNA/DNA, DNA/RNA duplexes so
that hybrid can occur at temp below 65- 75 C (normal temp for in situ to work, but causes
trouble with morphology) with 50% formamide in mix.
Dextran Sulfate: strongly hydrated, macromolecules have no access to
water- increases hybridization rates by apparently increasing probe
concentration.
Other variables;
Probe length - increase probe length = increase renaturation rate
Probe conc. - increase probe concentration = increase reanneal rate