IN SITU PROTOCOL 1 OF 10
Making RNA Probes
Step 1: Digest Plasmid
Plasmid
____  (5ug/conc. of gene)
10X Buffer
15 ul
Enzyme (Kpn1)
dH2O (no DEPC)
2 ul
Fill up to 150 ul
1. Incubate at 37˚C for 1.5 hrs in water bath
2. Remove and clean rxn using mini-Elute Kit (purple columns by Quaigen  works best)
a. Add 5 times volume of PBI Buffer to rxn mixture  750 ul
b. Transfer to purple column (max volume at 700 ul)
c. Centrifuge at 12 rpm for 1 min; repeat steps b-c until all volume is
transferred
d. Clean column with 750 ul of PE Buffer (make sure box is marked for
addition of ethanol)
e. Centrifuge and discard flow through
f. Centrifuge for an additional minute and put column in new RNase free
micro-centrifuge tube
g. Elute with 10 ul of EB Buffer
3. Run 1 ul on 1% TAE gel to visualize digest; make sure to also run a non-digested
sample for comparison
Step 2: Transcription of probe
Plasmid
2 ul (use 2 ul of the eluted 10 ul from previous step  should be 1 ug)
10X Buffer
2 ul
Enzyme mix
2 ul
DIG label
2 ul
DEPC H2O
12 ul (To fill up to 20 ul)
1. Incubate at 37˚C for 1.5 hrs in water bath
2. Remove and clean rxn using mini-Elute kit
a. Add 5X volume of PBI to rxn mixture  100 ul
b. Transfer to purple column (max volume 700 ul)
c. Centrifuge @ 12 rpm for 1 min
Revised Nguyen 10-4-06
IN SITU PROTOCOL 2 OF 10
d. Clean column with 750 ul of PE Buffer (make sure box is marked for
addition of ethanol)
e. Centrifuge and discard flow-through
f. Centrifuge for an additional minute and put column in new RNase microcentrifuge tube
g. Elute RNA in 10 ul of DEPC H2O
h. Add 41 ul DEPC H2O to a total volume of 50 ul
3. Aliquot 1 ul into an 0.5 ml RNase free micro-centrifuge tube for gel running and
another for Dot Blot
4. Run 1 ul on a gel (1.5% TAE) usually run at 60 V for 2 hrs for good separation
of ladder (RNA ladder = millennium marker)
5. Store in -80˚C freezer until use
Revised Nguyen 10-4-06
IN SITU PROTOCOL 3 OF 10
Dot Blot for Concentration of Probe
Purpose: A dot blot is done on probe after it has been transcribed in order to calculate the
quantity you have.
Safety: RNA-free  wear gloves and lab coat at all times. ONLY pour ~10 ml of each soln in
RNase treat box at a time so soln won’t overflow
Method: All steps take place on shaker and remember to thaw out solns prior to wash!
1. Make serial dilutions (use 5 - 1.5 ml micro-centrifuge tubes): Take correct ml from probe
and follow arrows to dilute
1ul
2ul
PROBE
Dilution:
2ul
Tube #1
Tube #2
4ul RNA Buffer
2ul RNA Buffer
5X
10X
5ul
5ul
Tube #3
8ul RNA Buffer
50X
Tube #4
Tube #5
5ul RNA Buffer
5ul RNA Buffer
100X
200X
2. Clean box with RNase ZAP and set up grid on nylon paper with pencil as example below:
Add 1 ul of each dilution to appropriate grid position
Control
Sample #1
Sample #2
So on…
5X
•
•
•
•
10X
•
•
•
•
50X
•
•
•
•
100X
•
•
•
•
200X
•
•
•
•
3. Bind RNA to filter using the spectrolinker  press “optimal crosslink” and then “start”
(don’t mess with time b/c time is already set)
4. Work filter with 2X SSC for 10 mins
5. Wash twice with PBT for 10 mins
6. Preincubate filter with PI buffer for 1 hr
7. Wash filter twice with PBT for 10 mins
8. Incubate filter DIG AP (1/5000 dilution) in PI buffer for 30 mins
a. For 10 ml DIG, add 200 ul of 1:100 DIG into 9800 ul PI
9. Wash twice with PBT for 10 mins
10. Dissolve 1 NBT tablet into 10 ml SDW (remember to use a new 15 ml tube each time
and to cover with foil  light sensitive); vortex to completely dissolve NBT tablet
Revised Nguyen 10-4-06
IN SITU PROTOCOL 4 OF 10
11. Incubate filter for 10 mins with NBT/SDW soln  cover RNase treated box with foil
12. Wash twice with PBT for 10 mins
13. Stop rxn with 4% PFA
a. Wash with 4% PFA for 5 min, take out with tweezer, dry nylon paper on paper
towel
b. Store nylon paper with sample to dry @ RT
Revised Nguyen 10-4-06
IN SITU PROTOCOL 5 of 10
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
1. 0.1% (v/v) DEPC-treated water store @ RT:
a. Fill bottle up to 1L with milli-Q water. Add 1 ml DEPC (stored in 4˚C
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.
2. 4% Paraformaldehye (4% PFA) stored @ 4˚C or -20˚C:
a. Measure 4 g Paraformaldehye (located in fridge). Add 10 ml of 10X PBS
(in situ). Fill up to 100 ml with DEPC-treated water. Put in water bath at
65˚C. Add several drops of 10M NaOH until most PFA dissolve.
3. 10X PBS (Phosphate Buffered Saline) stored @ RT:
a. 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.
4. 1X PBS stored @ RT:
a. 100 ml 10X PBS diluted with approx. 900 ml DEPC-treated water up to 1L.
5. PBT or PBST stored @ RT:
a. To 1L of DEPC-treated 1X PBS, add 1 ml of 20% Tween.
6. 20% Tween-20 stored @ RT:
a. To make 100 ml volume, add 20 ml Tween-20 and fill up to 100 ml with
DEPC-treated water.
Revised Nguyen 10-4-06
IN SITU PROTOCOL 6 of 10
b. To make 10 ml  add 2 ml Tween-20 and fill up to 10 ml with DEPCtreated water.
7. 20X SSC (for hybridization and washing) stored @ RT:
a. To make 1 L  weigh 263.6 g of SSC Buffer powder (located in molecular
biology cabinet) and fill up to 1 L with DEPC-treated water.
8. Hybridization Buffer w/o tRNA stored @ -20˚C: (no probe use here)
a. In a 50 ml tube, mix the following reagents: 32.5 ml formamide (this give
65% formamide final concentration), 12.5 ml 20X SSC/DEPC, 25 ul
Heparin (100mg/ml), 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.
9. Hybridization buffer with tRNA (Hyb w/ tRNA) stored @ -20˚C: w/ probe added fresh
a. With 50 ml of the hybridization buffer, add 500 ul of the yeast tRNA
solution (stored @ -20˚C).
b. Have 1% stock of tRNA, therefore, dilute 100X
c. Ex) to make 3 ml, add 30 ul of 1% stock tRNA
10. Heparin stored @ -20˚C:
a. Located in Dr. Scemama’s cabinet in small bottle above microwave.
b. Make a stock solution of 100mg/ml in DEPC-treated water. Store in
aliquots at - 20˚C.
11. AP Substrate Solution (NBT/BCIP tablets): Prepare fresh!
a. Add 1 tablet to 10 ml of DEPC-treated water. Dissolve well by vortexing
and cover with aluminum foil; or half a tablet with 5 ml of DEPC- treated
water
12. PI buffer (Pre-Incubation) stored @ -20˚C:
a. 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 ml with
PBT solution. Stored in 10 ml aliquots at - 20˚C!
13. Alkaline Phosphate (AP) buffer – DIG AP buffer – for DIG label: Prepare fresh!
a. Also known as Detective Buffer (10X  dilute to 1X w/ SDW)
Revised Nguyen 10-4-06
IN SITU PROTOCOL 7 of 10
b. 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.
14. 0.2X SSC stored @ RT:
a. To make 50 ml final volume, add 500 ul of 20X SSC and fill up to 50 ml
with DEPC-treated water.
15. 75% MetOH/PBT stored @ RT:
a. For 50 ml, add 12.5 ml PBT to 37.5 ml MetOH
16. 50% MetOH/PBT stored @ RT:
a. For 50 ml, add 25 ml PBT to 25 ml MetOH
17. 25% MetOH/PBT stored @ RT:
a. For 50 ml, add 37.5 ml PBT to 12.5 ml MetOH
18. Proteinase-K:
a. Stored at - 20˚C. Stock is 20mg/ml which is 2000X concentrated. Dilute 5
ul of 20 mg/ml stock into 10 ml of PBT.
19. Solution A
a. 75% hybridization mix + 25% 2X SSC at 70˚C
20. Solution B
a. 50% hybridization mix + 50% 2X SSC at 70˚C
21. Solution C
a. 25% hybridization mix + 75% 2X SSC at 70˚C
22. Anti-DIG antibody (pre-absorbed in PI buffer):
a. 2 ul anti-DIG antibody + 198 ul PI Buffer = 200 ul
b. 20 ul + 980 ul PI buffer = 1ml
c. To make 10 ml, add 200 ul + 9800 ul (or 9.8ml) PI buffer = 10 ml
i. This [10 ml] is PI – preabsorber anti-DIG antibody.
Revised Nguyen 10-4-06
IN SITU PROTOCOL 8 of 10
Prior to in situ:
o Fix embryos in freshly made PFA or “thawed” PFA 24 hrs after collecting
embryos at RT in glass vials until ready to be wash with MetOH or next step.
o Transfer embryos accordingly to each solns below
1. 1X
10 mins
30%Methanol/DEPC
2. 1X
10 mins
50%Methanol/DEPC
3. 1X
10 mins
70%Methanol/DEPC
4. 1X
10 mins
100% Methanol
5. 100% Methanol (embryos can be store here in -20˚C for further use)
IN SITU:
Day 1:
1. Transfer fixed embryos from glass vials into RNase free micro-centrifuge tubes
(in RNase free cabinet); turn on water bath and set it to 70˚C.
2. Wash embryos at room temperature using 1ml on shaker:
a. 1X
5 mins
75%methanol/PBT
b. 1X
5 mins
50%methanol/PBT
c. 1X
5 mins
25%methanol/PBT
d. 3X
5 mins each
100%PBT
3. Thaw out 4% PFA in water bath; take out when thawed.
4. Digest with Proteinase-K according to the stage of development at room temp.
12 somites to 24h
10 minutes
30-36hs
15 minutes
48+
20minutes
a. Add 0.5ul of 20mg/ml of stock proteinase-K into 1 ml 1X PBT (want final
concentration to be 10ug/ul)
b. REMEMBER: add 1ml 1X PBT into vials first before adding 0.5 ul of 20mg/ml
of stock proteinase-K
5. Aspirate (take out) proteinase-K immediately and refix with 4% PFA for 20
minutes at room temperature.
6. Prewarm pre-hybridization and hybridization+tRNA at 70˚C in water bath.
Revised Nguyen 10-4-06
IN SITU PROTOCOL 9 of 10
7. Rinse 5X in 1X PBT for five minutes each at room temperature.
8. Rinse in pre-hybirdization one time for five minutes at 70˚C in water bath.
9. Aspirate and transfer new pre-hybridization to micro-centrifuge tubes and
incubate in water bath for three hours at 70˚C.
10. Twenty minutes prior to finishing 3 hrs incubation, prepared probe in
hybridization+tRNA and incubate (heat shock) at 80˚C for ten minutes
11. Aspirate pre-hybridization solution and replace with probe+hybrdization+tRNA.
Incubate at 70˚C in water bath for at least 16 hrs.
Day 2:
1. Turn on Hybridization oven to 70˚C and do the following washes in Hyb. oven
a. 10min
75% hyb mix+25% 2X SSC
b. 10 min
50% hyb mix+50% 2X SSC
c. 10 min
25% hyb mix+75% 2X SSC
d. 10 min
100% 2X SSC
2. Wash 2X for 15 minutes each with 0.2X SSC at 70˚C in hybridization oven (dilute
2X to needed soln w/ DEPC-treated water)
3. Thaw out PI Buffer if needed or make PI Buffer
4. Wash 2X for 5 minutes each with 50% 0.2X SSC+50% PBT at room temp
5. Wash 2X for 5 minutes each with 100% PBT at room temp
6. Incubate embryos in Pre-Incubation (PI) buffer for 5 min at room temp. (PI
buffer= for 50 ml  49ml of PBT add 1ml sheep serum and 100mg Bovine S
Albumin in fridge stored at -20˚C)
7. Aspirate and replace with new PI buffer and incubate for 1 hour at room temp.
8. Incubate with a 1:5000 final dilution in PI PRE-ABSORBED anti-DIG antibody at
4˚C overnight on the rocker. (Dilute stock of antibody (1:100)  for 1.0ml, add
20ul of anti-body into 980ul PI buffer)
a. Remember: Add PI buffer first before adding antibody.
Revised Nguyen 10-4-06
IN SITU PROTOCOL 10 of 10
Day 3:
1. Wash 8X for 15 minutes each wash with PBT @ RT
2. Wash 3X for 5 minutes DIG AP Buffer (ALWAYS made fresh) at room
temperature. Use “DIG soln” box. Stock is 10X  dilute to 1X DIG AP Buffer
a. To make 40 ml  4 ml 10X DIG + 36 ml SDW
3. After the third wash with DIG AP buffer, make AP substrate (NBT/BCIP tablet)
solution at room temp and cover it in foil. Keep it in the dark on rotor.
4. After last wash of DIG AP buffer, transfer samples to wells in micro-plate with big
plastic pipette.
5. Remove all DIG AP buffer making sure to not expose embryos to air for more
then a few seconds
a. HINT: do one well at a time.
6. Add AP substrate (600 ul/well) to wells and cover microplate wells with aluminum
foil. Set wells back onto shaker.
a. HINT: Use new glass pipette during each new experiment
7. Monitor color development every thirty minutes
8. Stop reaction by washing with PBT quickly three times
9. Fix embryos in 4% PFA overninght.
Revised Nguyen 10-4-06