Semi-Automated Whole Mount in situ Hybridisation (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Melissa H. Little

Keywords

[‘in situ hybridization’]

Subjects

[‘Gene expression analysis’, ‘Molecular biology’, ‘Developmental biology’]

Release Date

2017-08-04

Abstract

Semi-Automated Whole Mount in situ Hybridisation protocol by the Little Group

Procedure

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

1. Dissect embryos of 9.5dpc (whole embryo), 10.5dpc (forelimb-hindlimb) and 12.5dpc(urogenital tract) in ice-cold PBS.

2. Immediately fix the tissues with 4% paraformaldehyde (PFA) overnight (16-18hours) at 4°C.

3. Wash the tissues twice with PBTX for 10 minutes each at room temperature.

4. Dehydrate with a series of 25% MeOH/75%PBTX, 50%MeOH/50%PBTX, 75% MeOH/25%PBTX washes for 20 minutes each, follow by two washes of 100% MeOH for 20minutes.

5. Store embryo tissues in 100%MeOH at -20°C.

Day 1- Cleaning, Re-hydration and Hybridisation (Blue System)

1. Place tray with nylon mesh baskets into the blue system and run the cleaning program.

2. Wash all the tubing in the blue system with 0.2M NaOH. (This is to maintain an RNase-free environment)

3. Wash all the tubing in the blue system with RO-H2O.

4. Carefully put the embryonic tissues into the baskets. Each basket contains 2x 9.5dpc, 3x 10.5dpc, 1 male and 1 female 12.5dpc, and 2 x12.5dpc/2d explants.

5. Start the re-hydration program of the blue system.

6. The samples are treated as follows:

1. Turn off robot and wash samples with 0.2% gluteraldehyde/4% PFA in PBTX in the fume hood at room temperature for 20 minutes.

2. Wash twice with PBTX at room temperature for 10 minutes each.

3. Remove baskets from tray and place baskets into a 48 well Cellstar plate containing 0.5ml pre-hybridisation solution in each well at 65°C for 2 hours.

4. Transfer baskets to a 48 well Cellstar plate with 0.5ml hybridisation solution at 65°C overnight. (Hybridisation solution = pre-hybridisation solution + ~0.2µg/ml DIG-labelled RNA probe.)

Preparation

1. Prepare the post-hybridisation washes and place at 65°C overnight.

Preabsorption of anti-DIG antibody

1. Prior to starting post-hybridisation washing, prepare the pre-blocking solution and pre-absorb the anti-DIG antibody Roche11093274910. (The pre-absorbed anti-DIG antibody can be kept at 4°C and recycled up to 3 times.)

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)

1. Remove the baskets from the 48 well plate and place in a tray filled with solution 1.

2. The robot will preheat the rocker with the samples to 65°C for 15 minutes.

3. The following stringency washes are performed:

Preparation

1. Prepare 0.1% BSA / TBTX at the end of day 2 and insert tubing into solution. The robot will start taking in 0.1% BSA / TBTX early morning of day 3.

Day 3 – Post-antibody washes and Development

1. The samples are washed as follows:

   | | | | | — | — | — | — | Post-Antibody   | RT     | 30 Minutes     | 0.1% BSA / TBTX | | Washes | RT | 30 Minutes | 0.1% BSA / TBTX | | | RT | 30 Minutes | 0.1% BSA / TBTX | | | RT | 30 Minutes | 0.1% BSA / TBTX | | | RT | 30 Minutes | 0.1% BSA / TBTX | | | RT | 10 Minutes | TBTX | The pH for NTMT decreases over time so it needs to be made fresh. The robot will pause for you to make up fresh NTMT and re-start it | | | | | — | — | — | — |                             | RT | 10 Minutes | TBTX | | | RT     | 10 Minutes     | NTMT | | | RT | 10 Minutes | NTMT | | | RT | 10 Minutes | NTMT |

2. Transfer contents of the baskets into 12 well plates with 1ml of BM purple substrate Roche11442074001 in each well.

   Bring the BM purple AP substrate to 15°C –20°C before using, no dilution is required but you may need to filter the solution to remove any precipitate.

3. Wrap the 12 well plates with aluminium foil and monitor samples for colour development approximately every 15-30 minutes.

   Note: Normally 3 control samples will be used to verify the success of the run. Controls are Wnt4 (strong), Wnt7b (medium) and Shh (weak). For Wnt4 and Wnt7b, generally the expression develops within 1 to1.5hours whereas Shh can take 25-30 hours.

4. When the colour has proceeded to the desired extent, stop the colour development by transferring the samples to a new 12 well plate containing distilled water. Depending on the extent of colour development, samples can be left overnight to continue developing or kept at 4°C to slow down the reaction.

   Note: If the samples have been over-developed, wash several times in PBS with 1% Triton X-100. This can be done for several days if necessary and will decrease the background issue.

5. Wash 3x with PBS at room temperature for 5 minutes each.

6. Fix samples with cold 4% paraformaldehyde at room temperature for 30 minutes.

7. Wash 3x with PBS at room temperature for 5 minutes each.

8. Store samples in 2ml Eppendorf tubes with PBS at 4°C.

9. Photograph as soon as possible. Use a petri dish with 1% agarose as a background.

Solutions & Volumes Required For W-ISH Using The Biolane Hti Robot

** Small Tray (20 Baskets)**

DAY1

Methanol     	70mL

PBTX            	350mL

PBT        	80mL

PBT/H2O2             50mL

Proteinase K      	50mL

Glut/PFA/PBTX   	40mL

PBT/H2O2

10mL  H2O2

40mL  PBT

Proteinase K (Roche3115879)

25uL  20mg/mL stock proteinase K

50mL  PBTX

Glut/PFA/PBTX

320uL  25% glutaraldehyde

48uL    Triton X-100

40mL   4% PFA in PBS

DAY2

Pre-Block     	50mL

Antibody      	 50mL

Solution1     	70mL

2xSSC          	70mL

2xSSC+CHAPS 80mL

0.2XSSC+CHAPS     80mL

TBTX            	180mL

TBTX+0.1%BSA  200mL

NTMT           	120mL

Pre-Block (100mL) N.B. If re-using antibody only require 50mL

2g BSA

10mL Sheep Serum

90mL TBTX

Antibody – Can store in fridge and re-use up to 3 times

15uL  anti-DIG antibody (Roche11093274910)

3mL   Pre-Block

18mg Embryo powder

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.

Solution 1

50mL Formamide

25mL 20x SSC

100uL Triton X-100

10mL 5% CHAPS

15mL water

2xSSC+CHAPS (80mL) 2xSSC+CHAPS (100mL)

8mL 20xSSC                10mL 20xSSC

1.6mL 5% CHAPS          	2mL 5% CHAPS

70.4mL H2O                88mL H2O

0.2xSSC+CHAPS (80mL) 0.2xSSC+CHAPS (100mL)

0.8mL 20x SSC             1mL 20x SSC

1.6mL 5% CHAPS          	2mL 5% CHAPS

77.6mL H2O               97mL H2O

TBTX+0.1%BSA

0.2g BSA

200mL TBTX

NTMT

12mL  1M NaCl

12mL  1M Tris.Cl pH9.5

6mL    1M MgCl2

120uL  Tween 20

90mL   H2O

Colour development solution

BM Purple (Roche 11442074001)

Solution Preparation for the W-ISH

Associated_Publications

• Rumballe BA, Chiu HS, Georgas KM and Little MH. Use of in situ hybridization to examine gene expression in the embryonic, neonatal and adult urogenital system. Methods in Molecular Biology: Kidney Development (commissioned book chapter).
• Rumballe B, Georgas K, Little MH. High-throughput paraffin section in situ hybridisation and dual immunohistochemistry CSH Protocols, 2008
• Georgas K, Rumballe B, Wilkinson L, Chiu HS, Lesieur E, Gilbert T, Little MH. Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney. Histochem Cell Biol. 2008 Nov;130(5):927-42. Epub 2008 Jul 11.

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium