30 Slide In Situ Hybridization Protocol

(Cathy Thut 8/19/98ãethanol dehydration version 1.0)

The protocol is performed in 1 set of 30 slide glassware (5 of the small dishes). The dishes plus 1 X 2L cylinder, 2 X 500 ml cylinders and a 1 or 2L flask should be baked at 180C for 11 hours before use. Place a stir bar in the flask and 2 of the dishes. Two additional dishes should contain slide racks.

Prior to the day of the experiment, you should prepare 3.5 L of DEPC water, 500 ml 10X PBS, 500 ml 20X SSC and 200 ml 1M Tris pH 9.5.

Tissue sections are cut at 12 um, collected on Superfrost plus slides and stored at ≠80C in slide holders placed inside tupperware filled with Drierite.

Day One--Hybridization:

Preparation:

Take the frozen slides out of the freezer 1 to 2 hours before you want to begin the procedure.

Make 2.1L 1X PBS in DEPC-water.

Make 300 ml 4% paraformaldehyde in 1X PBS. Heat on hot plate, with stirring, just until it goes into solution. DO NOT BOIL. Cool to room temperature in the hood. Pour into 1 glass dish.

Make hybridization solution (10 ml):

5 ml formamide (Gibco-BRL 5515UA)

2.5 ml 20X SSC pH 7.0

50 ul yeast RNA (50 mg/ml stock, Sigma R7125)

1.7 ml sheared salmon sperm DNA (3 mg/ml stock)

500 ul 100X Denhardtπs

250 DEPC water

Set up humidified hybridization chamber with Kimwipes on bottom. (This can be reused several times.)

12.5 ml 20X SSC

12.5 ml DEPC water

25 ml formamide

Hybridization procedure:

Label slides with numbers and probe names (use pencil) and place in rack.

Fix slides in 4% paraformaldhyde, RT, 10 min. Do not reuse the paraformaldehyde!

Wash 3 times in 300 ml 1X PBS, 3 min per wash.

Acetylate for 10 min (300 ml DEPC water, 3.5 ml triethanolamine, 0.75 ml acetic anhydride prepared immediately before use. Stir solution rapidly as you add the components).

Wash 3 times in 300 ml 1X PBS, 5 min. per wash.

Dehydrate through a graded series of ethanols made with DEPC water (30%, 50%, and 70% 3 min each then 2 X 100% 5 min each), 300 ml each. Make the ethanol solutions in the PBS dishes that have been rinsed with DEPC water.

Air dry 5-10 min.

Meanwhile, add DIG labeled probe to aliquots of the hybridization solution (each slide will need 150 ul probe/hyb mixture). Elaine resuspends the probes to 200 ng/ml in the hyb solution. Mix and heat to 80C for 4 min. Put on ice and bring to 4C before use.

Place slides in plastic slide holders (20 per holder). Add 150 ul probe to each slide and cover with a plastic coverslip (Oncor Inc., catalog #S1370-14), taking care to avoid air bubbles.

Place slide holders in humidified chamber and put in 60C oven O/N. Make sure slide holders are level.

Rinse glassware with d.i. water. Make 1 dish of 300 ml 5X SSC and 1 of 0.2X SSC in d.i. water. Put in 60C incubator O/N.

Day TwoãAntibody binding/color development:

Preparation:

Prepare B1 solution (1.2L, make 1.5L if you are making a new antibody stockã1.5L amounts are in parentheses):

13.92 g maleic acid (17.4 g)

10.5 g sodium chloride (13.1)

8.4 g sodium hydroxide (10.5)

water up to 1.2L (1.5L), pH to 7.5 using sodium hydroxide (2-5 ml 10M NaOH)

Prepare B2 (blocking) solution (300 ml):

Heat 300 ml B1 in a glass dish in microwave until nearly boiling.

Add 3 g blocking reagent powder to each, and stir with stirbar until dissolved. Bring to room temperature before use.

Prepare B3 solution (300 ml):

30 ml 1M Tris pH 9.5

6 ml 5M NaCl

1.5 ml 1M MgCl2

Prepare developing solution base (120 ml):

12 ml 1M Tris pH 9.5

2.4 ml 5M NaCl

12 g polyvinyl alcohol (70-100 kD)

water to 120 ml

Carefully microwave until PVA goes into solution (90C). Cool to RT.

Prepare 1 more dish with 300 ml 0.2X SSC at RT.

Post-hybridization washes:

Transfer a tray of slides from the humidfied chamber clean work surface. Place the slides with the coverslips into the 60C 5X SSC.

Once all the slides are in the 5X SSC, pull out the plastic coverslips, which should be very loose. Transfer the rack to the 60C 0.2X SSC and put at 60C for 1 hour.

Move dish with slides to room temperature for 5 minutes.

Transfer slide rack to 300 ml of room temperature 0.2X SSC for 5 minutes.

Blocking/ antibody binding:

Wash 5 mintues in 300 ml B1 solution.

Transfer rack to room temperature blocking solution for 1 hour.

Transfer rack to antibody solution for 1 hour (60 ul B. Mannheim anti-DIG antibody in 300 ml of B1 solution). This solution can be reused for several months. Spike it with 5 ul fresh antibody every two weeks or so.

Wash slides in B1 solution (300 ml), 2 X 15 min. at room temp.

Wash in B3 (300 ml) for 5 min. at room temp.

Color development:

Finish preparing developing solution (120 ml):

While stirring, add:

0.6 ml 1 M MgCl2

450 ul NBT

350 ul BCIP

to the cooled PVA solution.

Aliquot 16 ml of developing solution into 6 slide mailers and place 5 slides into each. The fifth slide should be turned backwards so the tissue does not rub against the side of the slide mailer.

Let incubate at 30C for 4 hours to 3 days. Check the intensity of the staining at the end of the day (4 hours or so) and then every day after.

When the color has developed sufficiently, rinse the slides in d.i. water 4 X 5 min to remove the PVA solution.

Transfer to 1X TE solution for 15 minutes.

Mount slides using 22X50mm slide covers using 2 drops of Aquamount solution. Dry flat O/N then seal edges with nail polish.