Whole-mount in situ hybridization - LNA probes (Wheeler lab)

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Dilution of new Exiqon probe, double ended, LNA

Delivered as a 1 nmol dried pellet

add 40 uL Sigma water (=25 mM)

Take 10 uL of this 25 mM solution and add to 12.5 mL hybridisation buffer (=20 nM)

Preabsorption

• Follow Day 1 of protocol with mixture of late stage embryos

• Remove probe on day 2 and repeat Day 1 approximately 4-6 times for a good preabsorption

Whole mount in situ hybridisation for LNA probes

'Day1'

Rehydration

• Washes: Place embryos in 24 well dish or 2ml Eppendorfs. Approx 10-20 per well/tube

X 1 in 75 % Methanol(H2O) 5 min at r/t on shaker

X 1 in 50 % Methanol(H2O) 5 min at r/t on shaker

X 1 in 25 % Methanol(H2O) 5 min at r/t on shaker

X 2 in PBST 5 min at r/t on shaker

Proteinase K

• Make proteinase K (from stock 10 mg/mL) 1:1000 in PBST

• Add proteinase K solution to embryos at r/t NO ROCKING

note: Approx time of incubation depends on stage of embryos:

St 10.5 = 1 min

St 12-16 = 2 min

St 16-20 = 3 min

St 20-25 = 4 min

St 25-30 = 5 min

St 30-33 = 6 min

St 33-36 = 8 min

St 36-40 = 18 min

St 40-45 = 20 min

• Wash X 2 in PBST

• Add 3.7 % formaldehyde(PBST) (15 mL = 1.5 mL formaldehyde + 15 m PBST) at r/t in fume hood for 20/45 min NO ROCKING

• Wash (5 min) X 2 in PBST at r/t in fume hood

Hybridisation

• Wash X 1 in 50 % hybridisation buffer(PBST) for 10 min at r/t on shaker

• Wash X1 in 100 % hybridisation buffer for 10 min at r/t on shaker

• Wash x 1 in 100 % hybridisation buffer for 4 hours at hybridisation temperature on shaker

Note: Hyb temp differs depending on probe

• Prewarm probe in hyb buffer to hybridisation temperature

• Add probe and leave o/n at hybridisation temperature on shaker

Day 2

Wash (unbound probe)

• Remove probe and replace in tube at -20˚C DO NOT DISCARD PROBE

• Wash X 1 in 100 % hyb for 10 min at hybridisation temperature on shaker

• Wash X 2 in “wash solution” for 15 min at hybridisation temperature

Blocking and anibody binding

• Wash X 1 in 50 % MABT (50 % wash buffer) for 10 min at hybridisation temperature on shaker

• Wash X 2 in 100 % MABT for 30 min at r/t on shaker

• Wash X 1 in 2 % BBR in MABT(2 % BMB) for at least 1 hr at r/t on shaker

• Wash X1 in 20 % Goat Serum(2 % BMB) for at least 1 hr at r/t on shaker

• Incubate in anti-Dig antibody (1/2000) made in 20 % Goat Serum(2 % BMB) o/n at 4˚C on shaker

Note: KEEP ANTIBODY (can be reused once or twice)

Day 3

Wash antibody

• X 3 in 100 % MABT at r/t

• X 6 in 100 % MABT at r/t for 1 hr on shaker

• X 1 in 100 % MABT o/n at 4˚C on shaker

Day 4

Colour reaction

• Wash X 2 in NTMT 10min at r/t on shaker

• Add colour mix (NBT/BCIP)(NTMT) until background develops at r/t on shaker with foil

Note 1) 4.5 μL NBT + 3.5 μL BCIP per ml NTMT

2) Stop if starts to turn pink - add PBST

• Wash TBST X 3 for 10 min

• Wash with PBST until signal can be seen at r/t on shaker

• Leave in cold room o/n in foil


  • COLOUR REACTION CAN BE REPEATED THE NEXT DAY
    • can dilute colour solution to observe signal that develops quickly

• Once desired colour achieved fix in MEMFA and store in Methanol. This can also help with removing background colour.

Hybridisation buffer (500 mL)

250 mL Formamide

32.5 mL 20 X SSC (pH 5 with citric acid)

25 mL 10 % CHAPS

10 mL 10 % Tween-20

5 mL 0.5 M EDTA (pH 8)

1 mL Heparin (50 mg/mL)

1.25 mL tRNA (yeast) (200 mg/mL)

Make up to 500 mL with DEPC H2O

Wash solution (500 mL)

250 mL Formamide (fume cupboard)

25 mL 20 X SSC (pH 5 with citric acid)

5 mL 10 % tween-20

Make up to 500 mL with DEPC H2O

MABT (500 mL)

100 mL 5 X MAB in cold room

395 mL d. H2O

5 mL 10 % tween-20

2 % BMB (50 mL)

10 mL 10 % BBR(MABT)

40 mL MABT

20 % Goat Serum(2 % BMB) 50 mL

10 mL 10 % BBR(MABT)

10 mL 100 % Goat Serum

30 mL MABT

NTMT (make fresh at every use) 50 mL

1.25 mL 2 M MgCl2

1 mL 5 M NaCl

2.5 mL 2 M Tris(HCl) pH 9.5

5 mL 10 % Tween-20

Make up to 50 mL with d.H2O

BBR 500 mL

50 g powder

500 mL DEPC H2O

TBST X10

80 g NaCl

250 mL 1 M Tris pH 7.5

2 g KCl

Make up to 950 mL with DEPC H2O and autoclave

Then add 50 mL Tween-20 and dilute to 5 X with DEPC H2O