In Situ Hybridization on Whole Mount Embryos 1 of 8
IN SITU HYBRIDIZATION ON WHOLE MOUNT EMBRYOS
-
All glassware must be baked overnight at 180˚ C
-
Orange caps/plastic rings should be treated with RNase away, rinsed with distilled
water and autoclaved.
-
Gloves MUST be worn at all times.
Solutions
-
0.1% (v/v) DEPC-treated water:
o Fill bottle approx. up to 1L with milli-Q water. Add 1 ml DEPC (stored in
fridge). Add a stir-bar. Let stir overnight. Autoclave next day. Do not
remove stir-bar. DEPC gets rid of RNase. Autoclaving gets rid of DEPC
(or) else DEPC will destroy the enzymes used in the experiment.
-
4% Paraformaldehye (4% PFA):
o Measure 4 g Paraformaldehye. Add 10 ml of 10X PBS (in situ). Fill
approx. up to 100 ml with DEPC-treated water. Dissolve in 10M NaOH.
Put in water at 65˚C.
-
10X PBS (Phosphate Buffer Saline):
o Mix 75.97g NaCL, 12.46g Na2HPO4 · 2H2O (Dibasic), 4.14g NaH2PO4 ·
H2O (monobasic, monohydrate). Dissolve in 800 ml DEPC-treated water.
Adjust the pH to 7.0 with 1M NaOH and 1M HCl. Do not put the pH probe
into the bottle (contamination!). Pour out some solution in a beaker.
Check its pH. If more or less, adjust with HCl or NaOH by adding a few
drops into the bottle. Pour some solution into the beaker. Check pH and
so on….until pH is close to 7.0. Sterilize by autoclaving.
-
1X PBS:
o 100 ml 10X PBS diluted with approx. 900 ml DEPC-treated water up to 1L.
-
PBT or PBST:
o To 1L of DEPC-treated 1X PBS, add 1 ml of 20% Tween.
-
Tween-20:
o To make 100 m volume, add 20 ml Tween-20 and fill up to 100 ml with
DEPC-treated water. Store at room temperature.
-
20X SSC (for hybridization and washing):
Original copy before 10/04/06
Unrevised extra info
12-14-06
In Situ Hybridization on Whole Mount Embryos 2 of 8
o Dissolve 175.3g NaCl and 88.2g sodium citrate in 800 ml of DEPC-treated
water. Adjust the pH with 1M Citric Acid to 6.0. Adjust the volume to 1L
and sterilize by autoclaving.
-
Pre-Hybridization Buffer (or) Hyb Buffer w/o tRNA:
o In a 50 ml tube, mix the following reagents: 25 ml formamide (this give
50% formamide final concentration), 12.5 ml 20X SSC/DEPC, 25 ul
Heparin (100mg/mc), 50 ul 20% Tween, adjust pH to 6.0 with 0.5M Citric
Acid (~920 ul) and fill up to 50 ml with DEPC-treated water.
-
Hybridization buffer with tRNA (Hyb w/ tRNA):
o To 50 ml of the pre-hybridization buffer, add 500 ul of the yeast tRNA
solution (stored @ - 20˚C Dr. Scemama’s baking oven). Keep at - 20˚C.
-
Heparin:
o Located in Dr. Scemama’s cabinet in small bottle above microwave.
o Make a stock solution of 100mg/ml in DEPC-treated water. Store in
aliquots at - 20˚C.
-
AP Substrate Solution (NBT/BCIP tablets): Prepare fresh!
o Add 1 tablet to 10 ml of DEPC-treated water. Dissolve well and cover with
aluminum foil.
-
PI buffer (Pre-Incubation):
o For 50 ml: add 1.0 ml sheep serum (stored in 1 ml aliquots at - 20˚C), 100
mg BSA (Bovin Serum Albumin fraction V_@ 4˚C), fill up to 50 m with PBT
solution. Stored in 10 ml aliquots at - 20˚C!
-
Alkaline Phosphate (AP) buffer – DIG AP buffer – for DIG label: Prepare fresh!
o In a 50 ml tube, mix 2.5 ml of 2M Tris base (pH 9.5), 2.5 ml of 1M MgCl2
(or MgCl2 hexahydrate), 1 ml of 5M Nacl, and 250 ul of 20% Tween.
Adjust to 50 ml with sterile dH2O.
-
0.05X SSC:
o To make 100 ml final volume, add 2.5 ml of 2X SSC (not 20X) and fill up
to 100 ml with DEPC-treated water.
-
60% MetOH/PBT:
o For 50 ml, mix 30 ml MetOH + 20 ml PBT
Original copy before 10/04/06
Unrevised extra info
12-14-06
In Situ Hybridization on Whole Mount Embryos 3 of 8
-
30% MetOH/PBT
o For 50 ml, mix 15 ml MetOH + 35 ml PBT
-
Proteinase-K:
o Stored at - 20˚C. Stock is 2000X and is of [ ????? ] 20mg/ml. Dilute 5 ul
of 20 mg/ml stock into 10 ml of PBT.
-
Solution A
o 50% 0.05X hyb mix + 50% 2X SSC at 70˚C
-
Anti-DIG antibody (pre-absorbed in PI buffer):
o 2 ul anti-DIG antibody + 198 ul PI Buffer = 200 ul*
o 20 ul* + 980 ul PI buffer = 1ml*
o To make 10 ml*, add 200 ul* + 9800(or 9.8ml) PI buffer = 10 ml

This [10 ml] is PI – preabsorber anti-DIG antibody.
Original copy before 10/04/06
Unrevised extra info
12-14-06
In Situ Hybridization on Whole Mount Embryos 4 of 8
IN SITU PROTOCOL
-
Wear gloves at all times! Shake on rocker.
Day 1
1. Put Samples in appropriate wells or microcentrifuge tubes according to age.
2. 5 minutes of each washes [done to rehydrate the embryos] at room temperature
w/ 300 ul of ~ 30 minutes [between washes, remember to wait 1-2 min to let
embryos settle down and then leave enough soln to cover all embryos in tube
before continuing to the next wash]:
a. 1X
1 mL 75% Methanol/PBT
b. 1X
1 mL 50% Methanol/PBT
c. 1X
1 mL 25% Methanol/PBT
d. 3X
1 mL 100% PBT washes : pH~7.0
3. Set a rocker in the 28˚C incubator. Take out as much of soln before digesting
reaction with Proteinase K. Set the samples immediately upright on the rocker
in the incubator according to digestion time below. The stock (20mg/ml) is stored
in -20˚C  final concentration needed is 10 ug/uL of Prot-K in PBT. Dissolve 5 ul
of stock into 10 ml of PBT.
[Increases target accessibility by digesting protein surrounding target nucleic
acid]. The extensive cross linking of proteins mask target nucleic acid; prot-K
makes holes into membrane.
Calculation:
5 ul into 10 ml
 5 ul into 10,000 ml
10,000 =2,000 fold  2,000 = 10 ug/ml (find concentration needed)
5
20*
Age
< 24 h
30-36 h
> 48 h
| Proteinase-K Digestion time
10 minuties
15 minutes
20 minutes
** THAW OUT 4% PFA (stored at 4˚C) in water bath.
Original copy before 10/04/06
Unrevised extra info
12-14-06
In Situ Hybridization on Whole Mount Embryos 5 of 8
4. Aspirate immediately and refix with 4% PFA for 20 mins.
Very importantPre-warm pre-hyb and Hyb (w/tRNA) solns @ 65˚C.
5. Rinse 5X 5 min each with PBT
6. Rinse in Pre-hybridization mix (=hybridization mix without yeast tRNA) one time
for 5 mins.
7. Incubate for a minimum of 3 hours @ 65˚C in Pre-hybridization mix (as in #6).
(#6 and#7 performed in pre-hybridization mix so as to prevent background
staining).
8. Hybridize with (probe) in hybridization mix with yeast tRNA at 65˚C overnight (at
least 16 hrs)
Calculations for hyb. Mix and probe
↓ (antisense)
SLα probe  ZFSLα – SP6 – DIG-As-probe
SLβ probe  ZFSLβ – SP7 – DIG-As-probe
SP6 and T7 are RNA polymerases.
Original copy before 10/04/06
Unrevised extra info
12-14-06
In Situ Hybridization on Whole Mount Embryos 6 of 8
Day 2
1. Turn on Hybridization Oven power and shaker, next to incubator, and set
temperature at 65˚C.
2. Solution A needs to be pre-warmed in water bath at 65˚C. The washes are done
@ 65˚C in Hybridizaiton Oven (between washes, let oven stand for 1-2 min to
warm up):
(diff. stringencies used to dissociate the nonspecific binding of labeled probes to
sequencing)
20X SSC stock solutions stored in - 20˚C  dilute stock to necessary solns for
washes:
Wash
Time
1X
50% Formamide + 50% 2X SSC (B)
30 mins
1X
100% 2X SSC
15 mins
2X
0.2X SSC
30 mins each
2X
100% PBT wash at room temperature
5 mins each
3. 5% sheep serum in PBT washes on shaker @ room temperature
a. 1X
5 mins
b. 1X
1 hours
4. Incubate in anti-DIG antibody (PI pre-absorbed)1 @ 4˚C (fridge) overnight (on
rocker).
Calculation:
 20 ul PAB(antibody)* (of 1 ml) + 980 ul of 1:100 PBT = 1 ml
******add soln directly into centrifuge tubes, pipette up and down to mix, DO NOT
Original copy before 10/04/06
Unrevised extra info
12-14-06
In Situ Hybridization on Whole Mount Embryos 7 of 8
touch embryos!
**Need DIG concentration of 1:5,000
However, we do not need of waste 10 ul of anti-DIG-antibody
let’s divide by 5.
1  (10/5)2 ul anti-DIG-antibody + (198/?) ul PI buffer = 200 ul*
To make diluted soln of 1 ml,
20 ul of the 200 ul + 980 ul PI buffer = 1 ml used
To make diluted soln of 10 ml, multiply by factor of 10:
(20 ul x 10) + (980 ul x 10) = 1 x 10 ml
2 200 ul + 9.8 ml = 10 ml Total final diluted volume!
1–
The anti-DIG-antibody conjugates to alkaline phosphatase and is visualized
with colometric alkaline phosphatase (AP) substrate such as NBT/BCIP (Nitroblue terazolium)/ 5-Bromo-4chloro-3-indolyphosphate)
Advantage of NBT/BCIP: good localization properties, high sensitivity and
stability of precipitates.
Original copy before 10/04/06
Unrevised extra info
12-14-06
In Situ Hybridization on Whole Mount Embryos 8 of 8
Day 3 : (No need of gloves!)
1. 8X 15 min each wash w/ PBT.  still part of Blocking reaction(?)
2. 3X 5 min DIG AP Buffer (made fresh always!) washes at room temperature.
3. Make AP substrate (NBT/BCIP tablet) solution @ room temperature, cover in foil.
Monitor color development.
4. STOP reaction by replacing the AP substrate solution with 5-6X PBT rinses (5
min each or rinse each wash immediately).
5. Fix embryos in 4% PFA.

Now, the fish is ready to be MOUNTED!

MOUNTING:
o For lateral view, use agarose.
o For dorsal view, use 80% glycerol/PBT
o Transfer fish/embryo from well into a petridish containing 80% glycerol.
Cut off the tail and remove the yolk out. Transfer to a slide using a pipet.
Put a coverslip on top gently, sticking it w/ Vaseline on 4 corners of
coverslip. Take a picture.
o Taking a picture

Make sure the dials are on 1,A,10/20 (when using 10X-, 50X-)

Pull •— on bottom left out when taking picture.

Top-left: pull rod out 50/50 to make slide visible. Pull it on “Photo”
when taking picture  will make stage black.

On Nikon Capture, take picture. Save on Nikon. Open pictures in
Adobe.
Original copy before 10/04/06
Unrevised extra info
12-14-06