|Title||Semi-Automated Whole Mount in situ Hybridisation|
Semi-Automated Whole Mount in situ Hybridisation protocol by the Little Group
Using the BioLane HTI Robot
The following method is partially based on the protocol by Wilkinson & Nieto Methods Enzymol. 1993;225:361-73 and adapted by Gemma Martinez and Brooke Gardiner.
BioLane HTI Robot
The BioLane HTI robot consists of two systems, a blue and a red system. Each system has 9 plastic tubes hooked into the robot labeled 1 to 9. The robot has been programmed so each tube has been assigned to a specific solution. By inserting the tube into the solution the robot will automatically take the required amount as generated by a script. The robot will gently rock the samples in a tray while the program is in progress.
Nylon mesh baskets are used by the robot to hold the tissues, each basket represents one probe. Three sizes tray are available with the BioLane HTI robot, each holds a different number of nylon mesh baskets. We routinely use the small (20 basket) tray.
Pretreatment of Embryos
Day 1- Cleaning, Re-hydration and Hybridisation (Blue System)
Preabsorption of anti-DIG antibody
18mg of embryo powder is placed in a 10ml tube with 10% sheep serum, 2% BSA in TBTX and 15µl anti-DIG antibody.
Incubate at 4°C for 3 hours or longer with gentle rocking.
Spin sample in a microfuge for 10 minutes, 4°C at 13000rpm.
Collect the supernatant and dilute it with 10% sheep serum, 2% BSA in TBTX.
Store the antibody at 4°C until use.
DAY 2 – Post-Hybridisation and pre-blocking (Red system)
Day 3 – Post-antibody washes and Development
Solutions & Volumes Required For W-ISH Using The Biolane Hti Robot
** Small Tray (20 Baskets)**
Proteinase K (Roche3115879)
Pre-Block (100mL) N.B. If re-using antibody only require 50mL
Antibody – Can store in fridge and re-use up to 3 times
Incubate this solution on rocker in cold room for 3 hours minimum and then spin down at 13 000 rpm for 10min. Collect the supernatant and add to 50mL pre-block solution before using in the in situ hybridisation.
2xSSC+CHAPS (80mL) 2xSSC+CHAPS (100mL)
0.2xSSC+CHAPS (80mL) 0.2xSSC+CHAPS (100mL)
Colour development solution
BM Purple (Roche 11442074001)
Solution Preparation for the W-ISH
|Semi-Automated Whole Mount in situ Hybridisation.pdf|
|Release Date||2017-08-04 16:03:07|
|Principal Investigator||Melissa H. Little, MCRI|
|Murdoch Children’s Research Institute|