In Situ Hybridization -NCS

Day 1.

1. 4% PFA in 1x PBS for 10 minutes (shaking).
2. Rinse in 1x PBST (0.1% Tween-20; Sigma) for 3X 10 minutes each.
3. Proteinase K digestion 10-20 ug/ml in 10mM Tris pH 7.5 for 10 minutes @ RT.
4. Rinse 2X 10 minutes each in PBST to stop Rxn.
5. Fix tissue 1X 5 minutes in 4% PFA/1x PBS
6. Rinse 3X 10 minutes each in PBST
7. Acetylated:

• PBS for 1 minutes (shaking)
• 100mM TEA (Triethanolamine) pH 8.0 for 2 minutes (shaking)
• 0.25% Acetic anhydride in 100mM TEA (pH 8.0) for 1 minute shaking.
• Dry slides for 30 minutes at 37 oC.

1. Hybridization solution (50% Formamide, 600mM NaCl, 10mM Tris-HCl [pH8.0], 1mM EDTA, 1x Denhardt’s solution, 0.25% SDS, 10% Dextran Sulfate, 200 mg/ml yeast RNA) Heated to 85 oC for 10 minutes.
2. Calculate the amount of desired probe you want to add to 200 ml of Hybridization solution and heat to 85 oC for 3 minutes.
3. Apply the Hyb/probe mix to appropriate slide and let go O/N with parafilm in Hybridization chamber with 50% formamide @ 60 oC. NOTE: This step should be done in the dark.

Day 2.

1. Wash slides in 2x SSC containing 50% Formamide @ 60 oC for 30 minutes.
2. Wash slides in 2x SSC twice @ 60 oC for 20 minutes each.
3. Wash slides in 0.2x SSC twice @ 60 oC for 20 minutes each.
4. Block section with TBST (Tris Buffered Saline w/ Tween-20) containing 10 % Normal Sheep Serum @ RT for 1 hour.
5. Incubate slides with alkaline phosphatase (AP)-conjugated anti–DIG antibody (1/1000) @ RT for 1 hour (In humidified chamber with TBST).
6. Wash slides for 10 minutes (shaking) in Buffer 1.

Buffer 1.

100mM Tris-Cl ( pH 7.5)

150 mM NaCl

1. Wash slides for 10 minutes (shaking) in Buffer 2.

Buffer 2.

100mM Tris-HCl (pH 9.5)

100mM NaCl

50mM MgCl2

1. Incubated with nitroblue tetrazolium salt (NBT) and 5- bromo-4 chloro- 3 indolyl phosphate toludinium salt (BCIP) @ RT O/N in humidified chamber with TBST.

Day 3.

1. Stop the Rxn with Buffer 3.

Buffer 3.

10mM Tris-HCl (pH 8.0)

1mM EDTA

Stock Solutions

DEPC H 2O

Add 500 ml DEPC per 1L dH 2O. Shake, let stand 1 hour, autoclave 20 minutes liquid cycle

8% Paraformaldehyde (100ml)

Heat ~80 ml H2O to 60 oC. Add 8.0 g paraformaldehyde. Turn off heat and stir briskly.

Add 1-2 drops 10N NaOH and stir until clear. Filter through #1 filter paper. Bring up to 100 ml and store at 4 oC.

10X PBS

9.94 g Na 2HPO 4 (dibasic) anhydrous

3.60 g NaH 2PO 4 (monobasic) anhydrous

75.972 g NaCl

Add to 800 ml DEPC H 2O. Bring up to 1L and check pH is 7.4. Add 500 ml of DEPC, shake, let sit 1 hour, autoclave 20 minutes liquid cycle.

10% Tween -20 (100ml)

Add 10 ml Tween -20 to ~85 ml DEPC H 2O and bring up to 100 ml . Stir to dissolve detergent.

100X Denhardt’s (for 50 ml)

2% polyvinyl pyrrolidone (1g)

2% Ficoll (1g)

2% Bovine serum albumin (1g)

Bring to 100 ml with DEPC H 2O, aliquot, and store at 20 oC

50% Dextran Sulfate ( 50 ml)

To 25 g dextran sulfate, add DEPC H 20 to 50 ml. Dissolve by shaking (this takes a while) Store at 4 oC.

20x SSC (1L)

175 g NaCl

88.2 Sodium Citrate Add to 800 ml dH 2O. Adjust pH to 7.0 with NaOH. Bring up to 1 L. Add 500 ml DEPC, shake, let sit, then autoclave.

0.2M Phosphate Buffer ( 2 L)

42.6 g Na 2HPO 4 (dibasic) anhydrous

13.8 g NaH 2PO 4 (monobasic) anhydrous

Add to 1800 ml dH 2O and bring up to 2 L.

pH to 7.2

100mg/ml Bovine Serum Albumin

Add 100 mg BSA to 1 ml dH 2O in microfuge tube. Vortex to dissolve. Store at 4 oC.

3% Triton X-100

Add 3 ml Triton X-100 to ~95 ml dH 2O. Stir to dissolve. Store at 4 oC.

1M Tris, pH 7.4 (1L)

140.4 g Tris HCl

13.4 g Tris Base

Add to ~850 ml dH2O. Adjust to pH 7.4. Bring up to 1L

5M NaCl

Add 292.2 g NaCl to ~800 ml DEPC H 2O and dissolve. Bring up to 1L. Add 500 ml DEPC, shake, let sit, then autoclave.

1M Tris pH 9.5 (500ml)

7.0 g Tris HCl

55.4 g Tris Base

Add to ~300 ml dH 2O. Adjust pH to exactly 9.5. Bring up to 500 ml.