Whole mount in-situ
Based on the Kelley lab - Ronna
1
Whole mount In Situ Hybridization
Plasmid DNA linearization
5µg
1.5µl
1.0µl
Plasmid DNA
Restriction enzyme (10U/µl)
10X buffer
Nuclease free water – up to 10µl
-------------------------------------------------------total volume
10µl
 Incubate at 370c at least 2hrs or overnight.
 Run 1µl of linearized plasmid + 8µl of Nuclease free water + loading buffer on 1%
agarose gel to ascertain plasmid linearization.
 Store plasmid in -200c.
Nucleotide mix
1/3 of the UTP should be labeled with digoxigenin. We use all of our labeled stock
solution, 25µl of UTP-digox/10mM. For an end concentration of 3.5mM we need 71.4µl
of total volume at the end. The solution has the following final concentrations:
10mM
10mM
10mM
6.5mM
3.5mM
GTP
ATP
CTP
UTP
UTP-digox
in 71.43µl of total volume
in 71.43µl of total volume
in 71.43µl of total volume
in 71.43µl of total volume
in 71.43µl of total volume
concentration of stock solution of ATP, GTP, CTP, UTP is 100mM, therefore we need
the following volumes:
GTP
7.14µl
ATP
7.14µl
CTP
7.14µl
UTP
4.64µl
Digox-UTP
25.0µl
Nuclease free H2O
20.37µl
---------------------------------------total
71.43µl
Whole mount in-situ
Based on the Kelley lab - Ronna
2
Probe synthesis
10X transcription buffer
INS (nucleotides)
Linearized plasmid DNA
RNAse inhibitor (100U/µl)
T3/T7/SP6 RNA polymerase (10U/µl)
Nuclease free water
2µl
2µl
2µg
2µl
2µl
up to 20µl total volume
1. Incubate @370c for 2 hours.
2. Run sample on 1% agarose gel: 1µl of RNA probe in 9µl of nuclease free water
and loading buffer.
3. Add 2µl DNAseI to RNA probe, incubate @370c, 15min.
4. Add 100µl TE, 10µl 4M LiCl, 300µl 100% ETOH, mix and store at -200c for RNA
precipitation.
Whole mount in-situ
Based on the Kelley lab - Ronna
Solutions:
PTW: 0.1% Tween-20 in PBS (1X)
Detergent Mix:
IGEPAL
500 µl
SDS
5 ml
deoxycholate
0.25 g
1 M Tris-HCl pH8
2.5 ml
500 mM EDTA pH8
100µl
5M NaCl
1.5 ml
DEPC H20
40.4 ml
-------------------------------------------------50.0 ml
Hybridization Mix:
Formamide
25.00 ml
1
SSC (20x pH5 w/citric acid) 3.25 ml
EDTA (0.5 M pH8)
0.50 ml
2
Yeast RNA (5mg/ml)
0.50 ml {final conc 50 µg/ml}
Tween-20 (10%)
1.00 ml
CHAPS (10%)
2.50 ml
Heparin (50 mg/ml)
0.10 ml {final conc 100 µg/ml}
Water
17.15 ml
----------------------------------------------------Total
50.00 ml
*Store at -20°C
5X MABT:
Maleic Acid (50mM)
11.6 g
NaCl (0.74M3)
8.7 g
Tween-20
11.0 ml
Water
185.0 ml
--------------------------------------------------Total
200.0 ml
*Add Maleic Acid and water, then pH to 7 w/ NaOH, then add other ingredients
NTMT:
5M NaCl
2.0 ml
1M TrisHCl
10.0 ml
2M MgCl2
2.5 ml
10% Tween-20
10.0 ml
Water
75.5 ml
-------------------------------------------------Total
100.0 ml
1
250ml 20SSC pH7 + 50ml 1M citric acid. For a 1M citric acid solution – take 29.4gr in 100ml of nuclease free water.
Other protocols use baker yeast tRNA at a 1mg/ml final concentration.
3
Take 30ml of a 5M solution.
2
3
Whole mount in-situ
Based on the Kelley lab - Ronna
NTMT-BCIP-NBT Mix:
NTMT
10 ml
BCIP(50 mg/ml in NTMT) 26.2 µl
NBT (75 mg/ml in DMF*) 33.7 µl
*Dimethyl Formamide
4% Paraformaldehyde in PBS
To make 250ml of 4% Paraformaldehyde
Wear disposable gloves throughout the procedure
1. Use a sterile 250ml Erlenmeyer flask.
2. Weigh out 10g of paraformaldehyde powder into flask.
3. Using disposable 50ml centrifuge tube, measure out 40ml of sterile water and put in flask.
4. Heat to 650c in an H2O bath.
5. Once solution has warmed to 650c, add 50µl of 10N NaOH, using a P200 tip.
6. Continue to swirl and heat solution in H2O bath.
7. Add 25ml of 10X PBS (Phosphate Buffer Saline), and 80µl of 6M HCl.
8. Bring solution up to 250ml with sterile H2O.
9. Check pH with litmus paper. Should range from 7.2 to 7.4.
10. Filter solution with 250ml filter bottle.
11. Label and place in cold room until ready to use.
4
Whole mount in-situ
Based on the Kelley lab - Ronna
I. Tissue Prep for ISH:
A. Dissection
1. Tissue is originally fixed in 4% PFA in PBS.
2. Dissect tissue in PBS.
B. Dehydration/Rehydration - ~1h
1. wash 2X for 5 min in PTW (optional – 2X 30min).
2. wash for 5 min in 50% Methanol in PTW
3. wash 2X for 5 min in 100% MeOH
*at this point tissues can be frozen at -20ºC for a few weeks
4. wash for 5 min with 75% MeOH in PTW
5. wash for 5 min with 50% MeOH in PTW
**optional: add a 25% step to the dehydration/rehydration
6. wash 2X for 5 min in PTW
II. Pre-treatments/Deproteination - ~1h40min
1. treat with 10 µg/ml proteinase K in PTW for 10 min4
2. remove proteinase K
3. carefully rinse with PTW 2X 5 min
**optional: incubate in Detergent Mix 2X 15 min, then rinse 2X 5 min PTW
4. post-fix in 4% PFA for 20 min
5. rinse with PTW
III. Hybridization – 1h30min until the o/n incubation
1. rinse in 1:1 PTW to hybridization mix and let cochleae settle
2. rinse in 1 ml hybrid mix and let cochleae settle
3. incubate horizontally at 65ºC on shaker in 1ml fresh hyb mix for 1 hour
4. add 1ml fresh pre-warmed hyb mix and 1µg (varies) DIG-labeled probe
5. incubate horizontally at 65ºC on shaker overnight (O/N)
---------------------------------------------End of day #1--------------------------------------------------------
4
Our proteinase K is at a 15mg/ml concentration. Add 0.7µl to 1ml of PTW.
5
Whole mount in-situ
Based on the Kelley lab - Ronna
6
IV. Post-Hybridization Washes
1. rinse 2X in pre-warmed hyb mix
2. wash 2X 30 min at 65ºC with 1.5ml pre-warmed hyb mix
3. wash 10 min at 65ºC with 1:1 hyb mix to MABT pre-warmed
4. rinse 2X with 1.5ml MABT
5. wash 15 min in 1.5ml MABT
**optional: incubate 1 hr with 1.5 ml MABT and 2% Boehringer Blocking Reagent (BBR), then add 2%
BBR to step 6 and step 7.
6. incubate 1 hour in MABT with 20% heat-treated sheep serum horizontally at 65ºC on shaker
7. incubate 3 hours RT or O/N at 4ºC on shaker in MABT, 20% sheep serum, and 1/1000 dilution APanti-DIG antibody
---------------------------------------------End of day #2-------------------------------------------------------V. Post-Antibody Washes
1. rinse 3X with MABT
-transfer tissue into glass vials
2. wash 2X 45 min with MABT at Room Temperature(RT) while shaking horizontally
VI. Histochemistry
1. wash 2X for 10 min in 5ml NTMT
2. incubate at RT in the dark while shaking with NTMT-BCIP-NBT mix
* purple color will develop (10 min- >3 days)
*change solution every few hours until color develops
3. remove solution when color is developed
4. wash for 10 min with 5ml NTMT
5. wash for 10 min with PBS
*can be stored in PBS at 4ºC
VII. Bleaching (Optional)
-used to minimize background color
1. warm cochleae to RT if originally at 4ºC
2. replace PBS with 70% or 90% EtOH
3. watch closely under microscope for background to start fading ( about 3 min)
4. when desired color is reached, quickly remove EtOH and replace with PBS