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Whole-mount in situ hybridization (Conlon lab) - Revision history
2024-03-28T19:59:09Z
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imported>Virgilio: Created page with "Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot] link to protocol document [http://www.unc.edu/~fconlon/Protocols/Whole-..."
2011-11-18T14:48:51Z
<p>Created page with "Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot] link to protocol document [http://www.unc.edu/~fconlon/Protocols/Whole-..."</p>
<p><b>New page</b></p><div>Protocol submitted by VGP from Conlon lab protocols [http://www.unc.edu/~fconlon/Protocols.htm#tropprot]<br />
<br />
<br />
link to protocol document [http://www.unc.edu/~fconlon/Protocols/Whole-mount%20in%20situ%20Hybridization%20Oct%202010%20PT.doc]<br />
<br />
<br />
Whole-Mount in situ Hybridization Protocol<br />
<br />
Updated 10/2/2010 PT<br />
<br />
<br />
Day 0 (Preparation of Digoxigenin-labeled probes)<br />
<br />
<br />
Linearize DNA with enzyme (5ug max, approx 3-4ul of ‘dirty’ miniprep). Check on gel. Clean using ph/chl extraction or using Probequant G50 microcolumns (GE Healthcare): place column in tube, break bottom seal and loosen lid slightly, spin 1 min 750g, discard tube. Place column in fresh epp, pipette 50ul (min vol!) rxn onto centre of compact bead column, spin 2mins 750g = clean linear DNA.<br />
<br />
<br />
Reaction: <br />
<br />
Linear DNA ~250-500 ng <br />
(5ul linear DNA from Probequant column as above)<br />
<br />
5X Promega Transcription buffer (10x if using NEB) 4 μl<br />
<br />
DIG RNA Labeling Mix (UTP), Roche 1277073 2 μl<br />
<br />
DTT (omit if using NEB buffer) 2 μl<br />
<br />
RNase Inhibitor (RNasIn) 1 μl<br />
<br />
RNA Polymerase (T7, T3, or SP6) 1 μl<br />
<br />
H2O to 20 μl total<br />
<br />
<br />
- 3-4 hr @ 37°C.<br />
<br />
<br />
DNase Digestion (optional)<br />
<br />
<br />
<br />
reaction 20 μl<br />
<br />
10X RQ1 DNase Buffer 3 μl<br />
<br />
RQ1 RNase-free DNase 7 μl<br />
<br />
Total 30 μl<br />
<br />
<br />
- 1 hr @ 37°C<br />
<br />
<br />
Probe Purification<br />
<br />
<br />
- Either repeat Probequant spin (make up rxn to 50ul before) or …<br />
<br />
- Use QIAgen RNeasy Kit:<br />
<br />
- Make up rxn to 100ul with RNAse free water<br />
<br />
- Add 350 μl Buffer RLT<br />
<br />
- Add 250 μl 100% EtOH<br />
<br />
- Apply to column, spin 10K 15 sec.<br />
<br />
- Transfer column to new tube<br />
<br />
- Add 500 μl Buffer RPE<br />
<br />
- Spin 15 sec, discard flow-through<br />
<br />
- Add 500 μl Buffer RPE<br />
<br />
- Spin 15 sec, discard flow-through<br />
<br />
- Transfer column to new tube <br />
<br />
- Spin 1 min.<br />
<br />
- Add 45 μl DEPC-H2O to column<br />
<br />
- Spin 1 min.<br />
<br />
- Add flow-through BACK TO COLUMN<br />
<br />
- Spin 1 minute<br />
<br />
- Check 2 μl on agarose gel<br />
<br />
<br />
<br />
Day 1 (Prehybridization)<br />
<br />
Rehydration of embryos:<br />
<br />
- Embryos should have previously been fixed in MEMFA for 2 hr @ RT or ON 4oC rocking, and stored @ -20oC MeOH (preferably ON before proceeding to enhance permeabilization)<br />
<br />
MEMFA<br />
<br />
MEM salts (10X) 1 ml<br />
<br />
Formaldehyde (37%) 1 ml<br />
<br />
DEPC-H2O to 10 ml<br />
<br />
<br />
<br />
<br />
10X MEM Salts<br />
<br />
MOPS 1 M 104.5 g<br />
<br />
EGTA 20 mM 3.8 g<br />
<br />
MgSO4 10 mM 1.24 g<br />
<br />
DEPC-H2O to 500 ml<br />
<br />
<br />
- Replace MeOH<br />
<br />
- 5 min @ RT, replace 0.5 volume with DEPC-H2O. Allow embryos to settle before beginning timer.<br />
<br />
- 5 min @ RT, replace 0.5 volume with DEPC-H2O<br />
<br />
- 5 min @ RT, replace 0.5 volume with DEPC-H2O<br />
<br />
- Replace all liquid w/ PBST<br />
<br />
<br />
DEPC-H2O<br />
<br />
DEPC 1 ml<br />
<br />
H2O 1 L<br />
<br />
(stir ON @ RT, then autoclave)<br />
<br />
<br />
PBST<br />
<br />
1X PBS (made w/ DEPC-H2O)<br />
<br />
0.1% Tween 20<br />
<br />
<br />
20X PBS<br />
<br />
NaCl 2.74 M 160 g<br />
<br />
KCl 54 mM 4.02 g<br />
<br />
KH2PO4 30 mM 4 g<br />
<br />
Na2HPO4 130 mM 18.46 g<br />
<br />
DEPC-H2O to 1 L<br />
<br />
(pH to 7.5 with HCL - autoclave)<br />
<br />
<br />
<br />
Transfer to PBST: (wash preferably in >3ml rocking on side)<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5 min @ RT in PBST<br />
<br />
<br />
<br />
Permeabilization of Embryos/Removal of Vitelline Membrane:<br />
<br />
<br />
For embryos hatched or with vitelline membrane removed<br />
<br />
<br />
- 15 min @ RT in Proteinase K (extend to 20-25mins for older >st40)<br />
<br />
Proteinase K (Sigma P-2308) – resuspend stock (18ug/ul) to 10 μg/μl. Add to embryos @ final concentration = 5 μg/ml (0.5 ul per ml PBST)<br />
<br />
<br />
<br />
For early embryos still in vitelline membrane (digests membrane)<br />
<br />
<br />
- 10 min @ RT in Proteinase K, Collagenase A, an Hyaluronidase. The membranes will come off during digestion or in the subsequent washes.<br />
<br />
Proteinase K (Sigma P-2308) – resuspend stock to 10 μg/μl<br />
<br />
Add to embryos @ final concentration = 10 μg/ml<br />
<br />
Collagenase A (Roche 103 578) – resuspend stock to 1 mg/10 μl<br />
<br />
Add to embryos @ final concentration = 2 mg/ml<br />
<br />
Hyaluronidase (Sigma H-4272) – resuspend stock to 20 U/μl<br />
<br />
Add to embryos @ final concentration = 20 U/ml<br />
<br />
<br />
Washes to Remove Proteinase K:<br />
<br />
- 5 min @ RT in PBST gently<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5 min @ RT in PBST<br />
<br />
<br />
Washes:<br />
<br />
- 5 min @ RT in 1ml 0.1M Triethanolamine<br />
<br />
0.1 M Triethanolamine<br />
<br />
0.93 g Triethanolamine<br />
<br />
30 μl 10 N NaOH (same as 10 M NaOH)<br />
<br />
50 mL DEPC-H2O<br />
<br />
- 5 min @ RT in 1ml 0.1 M Triethanolamine (2 mls per vial - DON’T REMOVE THIS SOLUTION)<br />
<br />
<br />
Neutralization of free amines: (prevents electrostatic interaction between probe and basic proteins)<br />
<br />
- 5 min @ RT, add 5 μl Acetic Acid Anhydride<br />
<br />
- 5 min @ RT, add 5 μl Acetic Acid Anhydride<br />
<br />
<br />
Washes:<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5 min @ RT in PBST<br />
<br />
Refixation:<br />
<br />
- 20-30 min @ RT in 4% Paraformaldehyde/PBST<br />
<br />
4% Paraformaldehyde<br />
<br />
paraformaldehyde 2 g<br />
<br />
10 N NaOH 1-2 drops<br />
<br />
microwave a few seconds to dissolve<br />
<br />
20X PBS (made w/ DEPC-H2O) 2.5 ml<br />
<br />
Tween 20 50 μl<br />
<br />
DEPC-H2O to 50 ml<br />
<br />
<br />
Washes:<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5 min @ RT in PBST<br />
<br />
- 5mins @ RT in 1:1 hyb:PBST<br />
<br />
<br />
Transfer to Hybridization Buffer<br />
<br />
- Add 1ml Hyb buffer.<br />
<br />
Hybridization Buffer (store -20oC)<br />
<br />
Formamide 25 ml<br />
<br />
20X SSC (made w/ DEPC-H2O) 12.5 ml<br />
<br />
Yeast Torula RNA 50 mg<br />
<br />
Heparin 5 mg<br />
<br />
50X Denhardt’s 1 ml<br />
<br />
Tween 20 50 μl<br />
<br />
CHAPS 50 mg<br />
<br />
0.5 M EDTA 0.5 ml<br />
<br />
DEPC-H2O to 50 ml <br />
<br />
(store @ 4°C for up to a few days)<br />
<br />
<br />
50X Denhardt’s<br />
<br />
Ficoll Type 400 1 g<br />
<br />
Polyvinylpyrrolidone 1 g<br />
<br />
BSA Fraction V 1 g<br />
<br />
DEPC-H2O to 1 L<br />
<br />
(store @ -20°C)<br />
<br />
20X SSC<br />
<br />
NaCl 3 M 175.3 g<br />
<br />
Na Citrate 0.3 M 88.2 g<br />
<br />
DEPC-H2O to 1 L <br />
<br />
(pH to 7.6 with HCl - autoclave)<br />
<br />
- ON @ 60°C (or appropriate temp) in Hybridization Buffer<br />
<br />
<br />
<br />
Day 2 (Hybridization)<br />
<br />
Hybridization:<br />
<br />
- Add 1-4 μg DIG-labeled probe to 1 ml Hybridization Buffer (~10 ul per ml)<br />
<br />
- denature probe/hyb 5 min @ 75°C<br />
<br />
- aspirate pre-hyb buffer from embryos and add the 1 ml probe/hyb<br />
<br />
- ON @ 60-63°C<br />
<br />
<br />
<br />
<br />
Day 3 (Antibody Staining) – <br />
<br />
- Pre-warm SSC and MAB buffers to 60oC<br />
<br />
Removal of Probe:<br />
<br />
- remove old probe – store @ -20/-80°C for re-use<br />
<br />
- rinse w/ Hybridization Buffer<br />
<br />
- 10 min @ 60°C in 1 ml Hybridization Buffer<br />
<br />
<br />
SSC Washes:<br />
<br />
- 20 min @ 60°C in 2X SSC<br />
<br />
- 20 min @ 60°C in 2X SSC<br />
<br />
- 20 min @ 60°C in 2X SSC<br />
<br />
- 20 min @ 60°C in 2X SSC <br />
<br />
<br />
RNase Treatment: (optional – don’t do for low-expression genes)<br />
<br />
- 30 min @ 37°C RNase <br />
<br />
RNase<br />
<br />
RNase A (4 mg/ml) 5 μl/ml (20 μg/ml total)<br />
<br />
RNase T1 (20 U/μl) 0.5 μl (10 μ/ml total)<br />
<br />
<br />
Washes: (unnecessary if RNase treatment was skipped)<br />
<br />
- 10 min @ RT in 2X SSC<br />
<br />
- 10 min @ RT in 2X SSC<br />
<br />
<br />
High-Stringency Washes:<br />
<br />
- 1 hr @ 60°C in 0.2X SSC<br />
<br />
- repeat again for highly expressed RNAs or to decrease background staining (don’t perform for low expression RNAs)<br />
<br />
<br />
Transfer to Maleic Acid Buffer:<br />
<br />
- 15 min @ 60°C in 1X MAB<br />
<br />
5X MAB<br />
<br />
Maleinic Acid 0.5 M 58.04 g<br />
<br />
NaCl 0.75 M 43.83 g<br />
<br />
DEPC-H2O to 1 L<br />
<br />
(pH to 7.5 w approx 9g NaOH pellets + additional NaOH solution)<br />
<br />
- 15 min @ RT in 1X MAB<br />
<br />
<br />
Blocking:<br />
<br />
- 60 min @ RT in 1 ml Blocking Buffer<br />
<br />
Blocking Buffer<br />
<br />
1X MAB 80% 8 ml<br />
<br />
Heat-inactivated lamb serum 20% 2 ml<br />
<br />
BMBR 2% 0.2 g<br />
<br />
Total 10 ml<br />
<br />
<br />
To heat-inactivate lamb serum: 1 hr @ 60°C, spin 15 min @ 14,000 RPM, store @ -20°C (SPIN BEFORE USE ABOVE)<br />
<br />
<br />
Antibody Incubation: (Fab Fragments, 1/2000 dil. Anti DIG AP Fab Fragments Roche 1093274)<br />
<br />
- Add 0.5ul/ml antibody to fresh blocking buffer.<br />
<br />
- Remove old blocking buffer, add antibody+blocking buffer onto embryos.<br />
<br />
- ON @ 4° C (rocking + standing).<br />
<br />
<br />
<br />
Day 4 (Removal of Antibody)<br />
<br />
<br />
Washes:<br />
<br />
- rinse in 1X MAB<br />
<br />
- 1 hr @ RT in 1X MAB<br />
<br />
- 1 hr @ RT in 1X MAB<br />
<br />
- 1 hr @ RT in 1X MAB<br />
<br />
- 1 hr @ RT in 1X MAB <br />
<br />
- ON @ 4° in 1X MAB (repeat washes if background high)<br />
<br />
<br />
<br />
Day 5 (Alkaline Phosphatase Staining)<br />
<br />
Transfer to Alkaline Phosphatase Buffer:<br />
<br />
- 5 min @ RT in AP Buffer<br />
<br />
<br />
AP Buffer<br />
<br />
1 M Tris pH 9.5 100 mM 5 ml<br />
<br />
1 M MgCl2 50 mM 2.5 ml<br />
<br />
0.5 M NaCl 100 mM 10 ml<br />
<br />
Tween 20 0.1% 50 μl<br />
<br />
Levamisol 5 mM 60 mg<br />
<br />
ddH2O to 50 ml<br />
<br />
- 5 min @ RT in AP Buffer<br />
<br />
Staining:<br />
<br />
- Add 1-2 ml BM Purple AP Substrate (Roche 1442074)<br />
<br />
- KEEP IN THE DARK UNTIL BLEACHING STEP BELOW TO AVOID BACKGROUND STAINING!!<br />
<br />
- Let stain anywhere from 1 to 8 hours until stain is at desired level<br />
<br />
- Rinse with PBS<br />
<br />
- 5 min @ RT in PBS<br />
<br />
- 5 min @ RT in PBS<br />
<br />
- 5 min @ RT in PBS<br />
<br />
- 1hr @ RT in MEMFA<br />
<br />
- 1 hr in MeOH (can be stored @ 4°C here)<br />
<br />
<br />
Bleaching: <br />
<br />
(can be done prior to Day 1 although requires re-fixing in MEMFA and starting from scratch. Also PFA fixation after Prot K treatment may need to be extended to 40mins/1hr as embryos are much more delicate.)<br />
<br />
- ON @ 4°C in 5:1 MeOH/H2O2 (30% solution) under high light (with rocking and on a reflective background such as aluminum foil)<br />
<br />
- Transfer and store in MeOH<br />
<br />
<br />
Clearing:<br />
<br />
- If you wish to clear embryos for image-taking, clear in 1:2 Benzyl Alcohol/Benzyl Benzoate (which can be REUSED!), but embryos MUST be fully dehydrated in MeOH before placement in BA/BB<br />
<br />
<br />
Take Pictures and Publish in Prestigious Journal<br />
<br />
<br />
The End</div>
imported>Virgilio