In situ hybridization protocol

In situ hybridization protocol – Peng Huang Lab

Day 0
Fix embryos in 4% PFA
5’ wash in PBT __ __
2’ 100% MeOH __ __ __ __
-20C in MeOH (at least 2hr)

Day 1
5’ in PBT __ __ __ __
ProK (10ug/ml; 1:1000 from 10mg/ml stock) RT __ (no shaking)
[1’ for 2-4S; 3’ for 9-13S; 5’ for18-24hpf; 8’-10’ for 30hpf; 20’ for 48hpf; 1hr or longer for >3dpf]
Quick PBT wash __
5’ in PBT __
20’ 4% PFA RT (30m-1hr) __
5’ in PBT __ __ __ (split fish into different tubes)
2hr in HYB (RT) at 70C __
O/N (12-18hr) HYB (pre-heated) w/ probe (1:100 from probe stock with 5% dextran sulfate) at 70C __
 

Day 2
Following at 70C: prewarm solutions, 500ul per tube
5’ 100% HYB __
5’ 75% HYB:2xSSC __
5’ 50% HYB:2xSSC __
5’ 25% HYB:2xSSC __
5’ 2xSSCT __
20’ 0.2xSSCT __
20’ 0.1xSSCT __ __
Following at RT: 500ul per tube
5’ 75% 0.2xSSC:PBT __
5’ 50% 0.2xSSC:PBT __
5’ 25% 0.2xSSC:PBT __
5’ PBT __
Block 2hr in 2mg/ml BSA + 5% sheep serum in PBT (>750ul per tube) on rocker at RT __
O/N at 4C in 150 ul Ab [sheep anti-digoxygenin 1:5000] in blocking solution on shaker __
 

Day 3
Following at RT on a rocker:
Quick wash in PBT __
25’ PBT (>750ul per tube, shake at RT) __ __ __*
5’ NTMT (400ul/tube) __ __ __
Develop with NBT/BCIP [33.75ul NBT+ 35ul BCIP in 10ml NTMT]: 400ul/tube RT or 32C __
Check after 30m, Stop reaction with PBT 5’ __ __ __ __ (make sure to wash sufficiently)
300ul 25%Glycerol:75%PBS __
300ul 50%Glycerol:50%PBS __
300ul 75%Glycerol:25%PBS __
* can be longer

Notes:
50ml NTMT (make fresh, scale it down if necessary):
5ml 1M Tris PH9.5
1ml 5M NaCl
2.5ml 1M MgCl₂
250ul 20% Tween20 (or 50ul Tween20)
To 50ml with H₂O