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