Visualization of Gene Specific Transcriptional Activity bv
In Situ Hybridisation.
Cleaning of microscope slides
1. Dip slides in 10% HCL/70% Ethanol for 2 min.
2. Wash slides in distilled water followed by 95% ethanol.
3. Wrap slide racks in alufoil.
4. Dry slides in oven at 150 0C for 5 min and allow to cool to
room temp.
5. Slides are now clean and "RNase free", ready to use
for coating.
Coating of the slides
Inappropriate coating may lead to loss of tissue samples from
the slides. Either TESPA (3-
aminopropyl-triethoxysilane, Sigma Cat. No. A-3648) or poly-L-lysine
(Sigma Cat. No. P- 2636)
can be used as an appropriate coating in order to fix the samples
tightly to the slides. The choice
of coating depends on whether or not the sample has already been
fixed before mounting on a
slide. TESPA coating of slides is very useful when working RNase
free is a prerequisite.
Forexample, when there is no fixation step before further processing
of the slides, which will be
the case when cutting sections from in wax embedded biological
samples.
Poly-L-lysine is usable for various purposes however
it doesn't garantee RNase free slides.
Another possibility is to use already polylysine coated slides
(Sigma diagnostics Cat. no.P0425).
TESPA coating
1. Dip slides in 2% TESPA in acetone for 10 sec.
2. Wash twice with acetone followed by distilled water.
3. Dry slides at 42 0C overnight.
Poly-L-lysine coating
20 mg/ml Poly-L-lysine stock soln. (20x) store at 4 0C. For useage
dilute this stock soln. In sterile
water till lmg/ml (DEPC treatment not nessesary), filter through
0.2 mm S&S filter.
1. Pipette 1.0-1.5 ml of PLL soln. onto a clean slide.
2. Spread this PLL equally over the surface of the slide using
a small paintbrush.
3. Mark the coated slide in such a manner that it will be easy
to recognize the coated side.
4. Cover the slide rack with alufoil and dry overnight at roomtemp.
5. Use these newly PLL-coated slides within several weeks.
Preparation of biological samples/material
We have prepared fetal liver, thymus, bone marrow, embryonic
peripheral red blood cells (still
nucleated) and a variety of tissue cultered cells for in situ
hybridization. Probably many other
tissue types can be prepared in a similar way.
1. Dissect out the target tissue and place it in PBS at roomtemp or 37 oC.
Example: fetal liver
| (gestational age) | 11.5 | 12.5 | 13.5 | 14.5 |
| volume PBS (ml) | 40 | 100 | 150 | 200 |
2.Disruption of the target tissue can be done in two ways:
by repeated pipeting using a yellow tip, keep the tip against
the bottom of the tube, until it looks like a homogenous cell
suspension or by using a cell homogenizer. Adult or older embryonic
tissues (more than 15 days of gestation) are difficult to disrupt
because they are tougher than younger embryonic tissue (younger
.than 15 days of gestation), homogenizing with a tissue homogenizer
might be of more help. Dilute embryonic blood and bone marrow
with approx. an equal volume of PBS and resuspend by repeated
pipeting to prevent clotting.
3.Apply 20 ml samples onto the coated slides in the shape of a
circle with a diameter of about 1cm. Placing multiple spot per
slide will make it possible to apply different sets of probes
on the same slide.
4. Allow the cells to settle down and attach to the glass for
1-2 min.
5.Place glass slide in the fixation solution for 20 min at room
temp. Fixation solution is 4% formaldehyde/5% acetic acid/saline
(8.5 g/l NaCl).
6. Wash slides three times 5 min. in PBS at room temp.
7. Rinse slides several min in 70% ethanol at room temp.
8.Store the slides in 70% ethanol at -20 0C till further usage.
Stored slides can be used up to several weeks.
Preparation of cultured cells for in situ hybridization
1. Take 1.5 ml of a confluent cell culture in a microfuge tube
(approx. 1-2x10 6 cells).
2. Spin down at 1500-2000 rpm for 1-2 min in Biofuge at room temp.
3. Wash cells in 500 ml PBS at room temp.
4. Repeat step 2.
5. Resuspend cells in 200-300 ml PBS (total approx. 1-2X10 7 cells)
6.Spread 20 ml aliquots of cell suspension on coated slides as
1 cm circles (approx 2x10 6 cells/spot)
7. Allow the cells to attach to the glass for 1-2 min.
8. Fixation and further treatment of the slides are as mentioned
above for the fetal liver cells.
Alternatively, the tissue culture cells can be grown on coated
slides, washed briefly in PBS
and from then on be fixed and treated as mentioned before. This
has several advantages: it is
faster, more gentle for the cells and less laborious. However
traces of serum on the slides may
result in relatively high background, therefore wash the slides
properly in PBS before fixation.
Pretreatment
The tissue samples are subjected to several pretreatment steps
before the hybridization probe is
applied. The use of pre-treatments is improve signal and reduce
background but must be
balanced by the desire to preserve the cellular structure. It
is advisable to ascertain the digestion
time when proteinases like pepsin or proteinase-K are used. Most
probably, each tissue or cell
type in combination with a particular fixation procedure needs
its own specific proteinase
treatment (concentration and treatment time) in order to gain
the best results and preservation of
structure. This has to be tested empirically.
The procedure underneath is optimilized for embryonic peripheral
bloodcells and disrupted fetal
liver cells.
1. Place slides in fresh 70% ethanol at room temp for 5 min.
2. Rinse slides in tris/saline (0.1M Tris-HCl ph 7.5; 0. 15M NaCl)
to remove ethanol.
3.Proteinase pretreatment: 0.1-0.01% pepsin (sigma) in 0.01M HCl
p112, 37 0C for 3-5 min (for fetal liver and embryonic blood,
0.01 % pepsin is enough).
4. Rinse in water briefly (1 min will do).
5. Postfix: 3.7% formaldehyde/PBS at room temp. for 5 min.
6. Wash for 10 mm with PBS
7.Dehydrate: 3 min washes in 70% EtOH, 90% EtOH and finally twice
in 100% EtOH, respectively. All ethanol solutions are made fresh
from a absolute ethanol and DEPC treated dH2O and are not reused
for following experiments.
8. Air dry and use these slides the same day for hybridisation.
Hybridization and washing of samples.
Many factors can affect the amount of signal and non-specific
background. The following factors
should be taken into consideration when results are unsatisfactory:
type and length of probe,
probe concentration, hybridization conditions (Temp, constitution
of hyb. soln., etc) and
stringency of washing. One should keep in mind that this protocol
is specifically written for the
use of oligonucleotides as probe therefore the above mentioned
factors should be revised when
different type of probes are used.
9.Put 24 ml of probe mix onto the slide when 24x50mm coverslips
are used or 12 ml probe mix when 24x24mm coverslip are used. Cover
the hybridization mix with a cleaned coverslip, do this gently
to avoid trapping of air bubbles. Coverslips are cleaned by briefly
rinsing them in water followed by absolute ethanol and subsequent
air drying.
10.Hybridize in a humidified chamber at 37 0C overnight. A humidified
chamber is made by placing paper towels on the bottom of the chamber
wetted with 25 % formamide, 2xSSC (similar conditions to hyb-mix).
HYBRIDIZE OVERNIGHT
11.Next day. Wash for 5 mins at 37 0C in 2xSSC (no formamide!)
to remove the coverslips; i.e. they should easily fall off without
damaging the cells.
12. Wash three more times with 2x SSC for 10 mins each, at 37
0C.
Choice of probe:
Our initial purpose for the use of the in situ hybridization
technique was to detect primary
transcripts of the different human beta globin genes. It is possible
to discriminate primary
transcripts from fully matured messenger by the use of intron
specific sequences as hybridization
probe. The choice of oligonucleotides as probe has several advantages.
Obviously,
oligonucleotides are small and therefore have an optimal penetration
capacity which also reduces
the need for excessive pretreatment and further allows the usage
of simple hybridization mixes
and low hybridization temperatures because of the low melting
temperature of the duplexes
formed. A recent study on the influence of the position and number
of biotin moieties per
oligonucleotide on the detection threshold and hybrid stability
has shown that relatively large
oligos (33 to 50 bp) with 3-biotin moieties labelled at both ends
and at the internal (middle)
position gave the most sensitive results (Eurogentec personal
comm.). Most of our
oligonucleotides are 50 basepair in length containing three hapten
moieties, at both ends and in
the middle, and are labelled with either biotin, digoxigenin and/or
DNP. However a larger variety
of haptens can be used as long as antibodies against them are
available.
Immunohistochemical detection of hybridized probe
An indirect approach for the detection of the primary transcripts
is used. In order to detect the
hapten moieties incorporated in the probes we applied a very sensitive
three layered
immunohistochemical detection system. Fluorescently labelled antibodies
are used because
fluorescence microscopy affords high resolution and a variety
of fluorochromes can be
distinguished in a single hybridization experiment. To give one
example of such a system:
Beta globin primary transcripts are hybridized with 4 digoxygenin
labelled 50 bp
oligonucleotides of which each contains three dig. moieties, are
detected by respectively,
sheep-anti-digoxinenin (monoclonal), rabbit-anti-sheep FITC labelled
(polyclonal) and finally
goat-anti-rabbit FITC labelled (polyclonal).
13.Rinse slides in Tris (0.1 M, pH 7.7), saline (NaCl, 0.15M)
containing 0.05% Tween-20 at room temp. for 5 min.
14.Preincubate cells with Tris/saline (0. iM Tris HCL pH 7.7,
0.15M NaCl) containing 0.5 % protein powder (Boehringer Mannheim
blocking reagent for elisa) at roomtemp. For 30-45 mins, use 100
ml soln. underneath 24x 50 mm cover slip.
15.Wash two to three times for 5 min at roomtemp. with Tris (0.1M
pH7.7), saline (0.15M NaCl) containing 0.05% Tween-20 for 5 mins
at R.T.
16.Apply antibodies (probe detection system) diluted as recommended
in Tris (0.1M, pH7.7), saline (NaCl, 0.15M) containing 0.5% protein
powder as blocking reagent (see step 14). Incubate at room temp.
for 30-45 mins. Use 100 ml antibody soln. per slide underneath
a 24mm x50mm coverslip. Incubate in a humid chamber, humidified
with tris (0.1M pH 7.7), saline (NaCl,0.15M).
Repeat washing acording to step 15 between each antibody application
of probe-hapten detect.
system
Repeat step 15 and 16 till all antibodies for detecting the hapten(s) of the immuno histochemistry detection system have been applied (one by one).
Finally end with:
17. Rinse slides for 5 min in tris/saline at roomtemp.
18.Dehydrate cells through 50% EtOH, 70% EtOH, 90% EtOH, and twice
100% EtOH and air dry as before.
19.Mount with DAPI/DABCO:Vectasheild (1:1) in glycerol based solution
and store in a dark and cold (4 0C) place. Apply 40 ml of the
mount soln. on the slides and place a 24mm x 50mm coverslip on
top with care, not to trap any air bubbles.
All solutions which are used in between fixation of the
target cells and washing after hybridization
has taken place, are made RNase free by treatment with DEPC followed
by autoclaving. Be
careful when working with DEPC, it is very toxic and volatile
(work in fumehood). DEPC will
fall apart into CO2 and alcohol (C4H10O). All the glass ware is
baked at 180 0C for 8 hrs.
NOTE. This protocol is writen specially for the use of oligonucleotides
as probes. When other
probes are being used several steps in the hybridization and washing
procedures have to be
ajusted.
Preparation of solutions for In Situ Hybridisation.
Hybridisation mixture
For the use of oligonucleotide probes:
(i) 25 % formamide (deionized)
(ii) 2x SSC
(iii) 200 ng/ml sheared salmon sperm DNA (carrier)
(iv) 5x Denhardt
(v) 1-5 ng/ml probe
(vi) 50 mM NaH2PO4/Na2HPO4 pH 7.0
(vii) 1 mM EDTA
Two stock soln. of oligonucleotide probes are made, one containing
250 ng/ml of each oligo and
the other containing 50 ng/ml. As probe concentration 0.5 ng/ml
is used and the probe soln is
added freshly on the day of use. The hybridization solution is
made as 9/10 part of the final hyb.
mix. On the day of use the hyb. mix is completed by adding probe(s)
and dH2O DEPC treated
(probe + water is 1/10 volume). The oligonucleotide probes are
disolved in dH2O DEPC treated
till 1 ml and stored at -70 0C.
1 M NaPO4 pH7 for Hybridisation mix:
Make 1 M soln with DEPC treated water of NaH2PO4*H20 (6.9 gr in
50 ml gives pH4) and
NaHPO4*2H20 (8.9 gr in 50 ml gives pH10),
Mix 5.7 ml NaHPO4 with 4m1 NaH2PO4 which gives a 1 M phosphate
buffer of pH7.
50x Denhardt's:
0.5 gr ficoll
0.5 gr polyvinylpyrolidone
0.5 gr BSA (pentax fraction V)
dissolve in DEPC treated dH2O and filter sterilize through nalgene
filter, store at -20 0C.
0.5 M EDTA, 5 M NaCl, 1 M MgC1, 20x SSC and 3 M NaAc are made
as usual, DEPC
treated and autoclaved
salmon sperm DNA:
disolve salmon sperm DNA till 10mg/ml in destilled water, shear
by 2x30 sec full sonication,
denature by 5' boiling, store in eppendorf tubes (lml) at -20
0C.
Formamide:
GIBCO BRL ultrapure Cat# 5515UA, store at -20 0C used as delivered.
DABCO/(DA)PI solution:
(i)Dissolve 1g DABCO (SIGMA) in 90 ml glycerol for 15-30 mins
at 60 0C.
(ii)Add l0ml of 1 M Tris pH 7.5.
(iii)Adjust to pH 8 with 5M HCL.
(iv)Cool to roomtemp.
(v)Make 0.02% azide (sodiumazide) or add 100 ml of 20% thermisol.
(vi) Add 15 m1 DAPI (lmg/ml stock in water, provides a blue background
stain) or either 100ml of
PI (lmg/ml stock in water, provides a red background stain).
(vii) Store in dark at 4 0C.
DAPCO - Vecta sheild mounting solution
(viii) Mix DAPCO/DAPI mount at a ratio of 1:1
Vectashield mounting medium for fluorescence:
Vector laboratories, Burlingame, CA 94010, order no.H-1000
DAPI:
4' ,6-Diamidino-2-Phenylindole, sigma D-9542 (10 mg)
DAPCO:
1 ,4-Diazabicyclo-[2,2,2])ctane, sigma D-2522, (25 mg)
Natriumazide: MERCK Art. 822335
Phosphate Buffered Saline (PBS):
lOx stock:
soln. 1: NaCl 200 g, KCl 5 gr in 1 liter of distilled water
soln.2: Na2HPO4*2H2O 72 gr, KH2PO4 10 gr in 500 ml distilled water
400 ml soln.1 + 100ml soln.2 + 500 ml distilled water = lOx PBS
DEPC treatment, autoclave.
2 M TrisCl:
dissolve 121.1 gr tris (ultrapure) in 300 ml DEPC treated destilled
water, bring the pH
down to 7.5 with concentrated HCl, volume up to 500ml with DEPC
treated distilled water.
The pH value of 2M tris is around pH10, in order to bring it down
to pH 7.5 around 64 ml of
concentrated HCl is needed.
lOx Saline:
dissolve 85 g NaCl in 1 liter (1.45 M) distilled water DEPC treatment
and autoclave.
Blocking reagent:
blocking reagent for elisa Boehringer Mannheim, cat no.1 112 589,
27 gram, dissolve this 27 g in
l00ml in dH2O (27% soln), aliquot in lml samples and store at
-20 0C This solution is 20x diluted
when used for antibody detection in insitu hybridisation experiments.
Diethyl Pyrocarbonate (DEPC):
Sigma cat no. D-5758 (be careful when using, very toxic)
always work in a fumehood. Add 100 ml DEPC to 500 ml solution
or dH2O, close the bottles
tightly and shake vigoursly. Incubate for several hours to overnight.
Autoclave the solutions.
Glassware do not have to be treated with water containing DEPC
as sometimes recommended.
Pepsin:
sigma P-7000, from porcine stomach mucosa (100 gr)
Make fresh soln. every time when doing a new in situ, dissolve
approx. 0.3 gr in 10 ml which is
then used as a stock solution for that day.
Poly-l-Lysine:
sigma P-2636, 100 mg.
Dissolve in 5 ml to make a 20 mg/ml stock soln., working soln.
is 1 mg/ml.
Dilute 20 mg/ml stock with dH2O till 1 mg/ml and filter steralize.
Formaldehyde:
Histologie Formaldehyde solution mm. 37%, free from acid,
MERCK cat no.3999, 1liter
(stabilized with about 10% methanol and calcium carbonate)