Tzahor Lab

In Situ

Protocol for RNA Whole Mount In Situ Hybridization

Contents: Day 0, Day 1, Day 2, Day 3, Day 4, RNA Whole Mount Double In Situ Hybridization, Solutions

♦ DAY 0 - Dehydration

Fixation and Dehydration with MEoH:

  • Fixation with 4% PFA was done over night at -40 (maximum).
  • In the morning replace to 1% PFA (not a must if you start dehydration straight away).

Dehydration

  • PBT 0.1% : 200ml PBSX1 (-/-) with 200ul Tween
Wash PBT 3X5°
25% MEoH 1X5°
50% MEoH 1X5°
75% MEoH 1X5°
100% MEoH 3X5°(in the 3rd time you keep the embryos)

Over night at -20°

♦ DAY 1 - Prehybridization and Hybridization

1. Rehydration  
  75% MeOH/PBT
50% MeOH/PBT
25% MeOH/PBT		 1X5°
1X5°
1X5° 
2. Bleaching [optional] - bleach if embryos are stage 19 and up.
  6% H2O2 in PBT for 1 hour. (e.g. 600 ul H2O2 in 10 ml PBT).
3. PBT 2X5°
4. Proteinase K   15° (1:1000 in PBT  from 10mg/ml stock)
  17°-18° for big embryos, 10° for small (st. 10) embryos.
5. Glycine (2 mg/ml in PBT)
(Warm for better solubility)		 1X10° // stops the proteinase k reaction.
6. PBT 2X5°
7. 4% PFA +25% Gluteraldehyde
(3968ul PBT + 32ul of stock 25%)		 1X20°-30°
8. PBT Rinse
-Pre warm PreHyb to 68°-
9. PBT  2X5°
10. 50% Prehyb mix/ 50 % PBT  1X10° - Pre-warmed and in bath
11. PreHyb >1h - Pre-warmed and in bath
12. Replace with exactly 2 ml PreHyb and add 5-10 ul of Probe
    O/N at 68° - Pre-warmed and in bath
  (Either Fluorecin or Digoxin labeled probes - amount depends on potency of probe)

Over night at 68° in warm bath

♦ DAY 2 – Post-hybridization washes, blocking, and antibody incubation

1. Prewarmed Solution X   1X5° Pre-warmed and in bath
2. Prewarmed Solution X 4X30° Pre-warmed and in bath
3. Prewarmed 50:50
Solution X/MABT		 1X10° Pre-warmed and in bath
4. MABT 1X5° RT with shake
  Transfer embryos into net, placed in 6 well. Transfer net to next well each wash
5. MABT  3X10°  
6. 20% goat serum in MABT >2h  
7. Anti-DIG 1:2000 (or Anti-Fluorecin 1:1500)   O/N at -4° with shake
  (In 20% goat serum in MABT). Cover with aluminium foil.

O/N at -4° with shake

♦ DAY 3: Post-antibody washes

1. Transfer to RT    
2. MABT 3X10° RT with shake
3. MABT 1X1 hour RT with shake – repeat until going home
4. Cover with aluminum foil and transfer to O/N shaking at -4°

O/N shaking at -4°

♦ DAY 4: Color development

1. Transfer to RT    
2. NTMT 3X10° RT with shake
3. Reaction mix: BCIP (1:1000) and NBT (1:1000) in NTMT – mix in hood!
Allow the reactions to develop at room temperature with gentle rocking. Throughout the development reaction, keep samples covered with aluminum foil to protect them from light and check them periodically to monitor the progress of the reaction.
4. Stopping the reaction:
NTMT
Rinse		  
5. TBS-T 3X10° RT with shake until embryos are clear
  Transfer to a glass vial
Replace PBS with PFA 4%, cover in aluminum foil and keep at 4°C.

♦ RNA Whole Mount Double In Situ Hybridization

After color reaction:

1. TBST O/N at -4° with shake
2. PFA 4% 1X30°
3. TBST 3X 5°
4. TBST 1X 30° at 65°
5. TBST 2X 5°
6. 0.1M Glycin-HCl (PH 2.2)  1X 15°
7. TBST 2X 5°
8. MABT 2X5°
9. MABT 2X30°
10. Blocking
(20% Goat Serum in MABT)		 2h
11. Antibody O/N

O/N at -4° with shake

♦ Solutions:

Pre-hyb 
50% formamide (use highest grade – deionized at 40C)
5X SSC, pH 4.5 (use citric acid to pH)
2% SDS 
50 mg/ml yeast tRNA
50 mg/ml heparin		 for 50 ml
25 ml
12.5 ml // from X20 stock
10 ml // from 10% bottle
250 ul // from sigma
250 ul // from 10 mg/ml stock
Complete with DDW-DEPC up to 50 ml
Solution X 
50% formamide (below the hood)
2X SSC, pH 4.5
1% SDS		 for 50 ml
25 ml
5 ml
5 ml
Complete with DDW up to 50 ml
MABT
Maleic acid 1M pH 7.5
NaCl
Tween 1%		 for 200 ml
20 ml
6 ml // from 5M stock
200 ul
Complete with DDW up to 200 ml
NTMT
NaCl
Tris-HCl 2M pH 9.5
MgCl2
Tween 1%		 for 200 ml
4 ml // from 5M stock
10 ml // from 20X stock
10 ml // from 1M stock
200 ul
Complete with DDW up to 200 ml