Hello,

I seem to be running into issues with my brain section in-situ hybridization. There is no signal present and there appears to be some sort of yellow crystaline deposites from the NBT/BCIP solution (Sorry for the poor quality image, a scope with a camera wasn't available at the time). Two slides were also prepared using 10 fold excess probe concentration, since we've been having issues getting any signal. The probes used were tested with a dot blot in a serial dilution on membrane exposed to NBT/BCIP, and appear to be of the desired concentration, about 100 ng/ul. DEPC treated water was used for all solutions.

Here's the protocol used on mouse brain sections pulled from a -80 freezer. The sections were prepared from brains embeded in OCT and cut with a cryostat.

- Air dry sections for a minimum of 20-30 min (up to 4-5 hours is fine).

II. Prehybridization and hybridization

Most of the remaining protocol is done in standard histology staining dishes and slide racks. The volumes listed here are for a 30-slide (one rack) experiment using 300 mL. of solution for each step.

- Fix the slides in 4% paraformaldehyde (in 1X PBS), RT, 10 min.

- Wash 3 times in 1X PBS – 3 min. per wash.

- Acetylate for 10 min. (295 mL still water, 3.5 mL triethanolamine, 0.75 mL acetic anhydride, prepared right before use- stir solution rapidly as you add components to the water.)

- Wash 3 times in 1X PBS – 5 min per wash

Make hybridization buffer (this is used for both the pre-hyb and hyb)

For 10 mL:

5.0 mL formamide

2.5 mL 20x SSC

50 uL of 50 mg/mL yeast RNA

1.67 mL of 3 mg/mL salmon sperm DNA

500 uL 100X Denhardt’s reagents

280 uL DEPC water

- Add 200 uL hybridization buffer to each slide. Cover slides with parafilm if necessary. Avoid air bubbles!Place in a humidified chamber (5X SSC and 50% formamide) and let sit at RT for 3-6 hours.

- Add digoxigenin-labeled RNA probe to hybridization mix and heat at 80°C for 5 min. We use the probe at 200 ng/mL. Cool on ice and then add 100 uL per slide. Cover with parafilm.

Incubate at 60-65°C for 16-24 hours in a air incubator. Place the SSC solution for the next day at 60°C overnight.

DAY 2

Prepare SSC solutions for washes:

0.2X SSC pH 7 make 600mL and split into two 300mL histology dishes.

5X SSC pH7 make 300mL

Heat the 5X SSAC and one of the 0.2XSSC dishes to 60°C (or to whatever the hybridization step was). Microwave both dishes 3-4 minutes, then equilibrate for 30 min – 1 hour in the 60°C incubator.

Prepare B1 solution: 1200 mL (make 1500mL if you are making a new antibody stock)

13.92 g maleic acid

10.5 g sodium chloride 8.4 g sodium hydroxide pH to 7.5 using sodium hydroxide (about 6.5-7mL of 5M NaOH) Make B2 (blocking) solution by adding 3 g of blocking reagent to 300 mL of pre-heated (65°C,