1. Removal of Paraffin

Caution: Perform the following steps under a fume hood.

1. Dewax the thin-sectioned paraffin-embedded slides with 100% xylene under the hood in glass Coplin jars for 10 min. Do not use plastic Coplin jars, as xylene melts plastic. Sterilize the Coplin jars in an autoclave before use.
2. Prepare four sterile glass Coplin jars with the following: 100% ethanol, 80% ethanol, 70% ethanol and 60% ethanol. Dilute the ethanol with RNase-free water.
   1. Transfer the slides to the sterile glass Coplin jar with 100% ethanol and soak them for 10 min. After 10 min, empty the glass Coplin jar and replace with new 100% ethanol. Soak the slides again for 10 min.
   2. Transfer the slides to the sterile glass Coplin jar with 80% ethanol for 1 min.
   3. Transfer the slides to the sterile glass Coplin jar with 70% ethanol for 1 min.
   4. Transfer the slides to the sterile glass Coplin jar with 60% ethanol for 1 min.

2. Pretreatment of Slides for Preparation of RNA Probe Hybridization

1. Perform the following washes at room temperature, unless specified otherwise. Perform room temperature washes on an orbital shaker at a slow speed. Use sterile slide mailers with room for up to five slides for the washing of the slides.
2. Transfer the slides to a sterile slide mailer with 18 mL of 1x phosphate buffered saline (PBS). Wash for 5 min at room temperature.
3. Pour off the 1x PBS, and immediately treat the tissue on the slides with proteinase K to enable probe penetration of the tissue. Add approximately 18 mL of proteinase K solution to the slide mailer (see Table 1 for working solution concentration). Incubate at 37 °C for 15 min. Do not shake.
   1. While the slides are incubating, prepare the prehybridization buffer (Prehybe Buffer). Place the Prehybe Buffer in a boiling water bath for 10 min, then cool on an ice bath for 5 min. For a recipe of Prehybe Buffer, see Table 1.
   2. Stop digestion of the proteinase K reaction by pouring out the proteinase K solution, and immediately add 18 mL of 0.2% glycine-PBS solution to the slide mailer. Let the slide mailer sit at room temperature for 5 min.
   3. Pour out the 0.2% glycine-PBS solution and add 18 mL of 2x saline sodium citrate (SSC) solution to each slide mailer. Wash the slides in the 2x SSC for 10 min at room temperature, with shaking at 100-150 rpm.

3. Prehybridization of Slides in Preparation for RNA Probe Hybridization

1. While the slides are being washed with 2x SSC, obtain a new sterile slide mailer and put approximately 18 mL of Prehybe Buffer into the slide mailer. Place the slide mailer into the hybridization oven at hybridization temperature.
   NOTE: The hybridization temperature will depend on the probe being used but usually ranges from 50-60 °C.
2. Once the 2x SSC wash is completed, pour out the 2x SSC, and place the slides within the slide mailer in the Prehybe Buffer in the hybridization oven for 1 h.

4. Hybridization of the RNA Probe

1. While the slides are incubating with the Prehybe Buffer in the hybridization oven, prepare hybridization-probe solution.
   1. Dilute each probe in hybridization buffer (0.5 µL of probe with 24.5 µL of hybridization buffer).
      NOTE: The recipe for hybridization buffer is found in Table 1. An example of probe preparation and concentrations can be found in Traylor-Knowles et al.¹¹.
   2. Place the diluted probe on an 86-90 °C heat block for 12 min, then cool for 1 min on ice.
2. Remove the slide mailer from the hybridization oven and individually remove slides from the slide mailer using sterile tweezers. Lay the slides flat on a paper towel, and carefully wipe off excess Prehybe Buffer around the tissue samples. Be careful not to touch the samples, and work quickly to prevent drying of the tissue samples.
3. Encircle the tissue with a PAP pen. Apply 25 µL of diluted probe solution with a pipette and cover the sample with a plastic coverslip.
   1. Place the samples in the slide moisture chamber with 4x SSC + 50% formamide solution in the bottom of the chamber.
   2. Once probes have been added to all the slides, place the slide moisture chamber into the hybridization oven for at least 24 h at a hybridization temperature of 50-60 °C. The incubation time can vary depending on the concentration of the probe but should be for a minimum of 24 hours.
4. After overnight incubation with the diluted probes, remove the slide moisture chamber from the hybridization oven.
   1. Remove coverslips from each of the slides, being careful not to displace the tissue. In a 1000 µL pipette, add 1000 µL of 2x SSC solution and gently wash the slide off.
   2. Place the slide in a sterile slide mailer with 18 mL of 2x SSC solution for 5 min at room temperature. Pour out the solution and replace it with 18 mL of fresh 2x SSC. Repeat the incubation again for 5 min at room temperature with gentle shaking.
   3. Pour out the 2x SSC solution. Add 18 mL of 1x SSC solution and wash for 5 min at room temperature. Pour out the 1x SSC solution and replace it with 18 mL of fresh 1x SSC. Repeat the incubation for 5 min at room temperature with gentle shaking.
   4. Pour out the 1x SSC. Add 18 mL of 0.5x SSC and wash for 10 min at 42 °C without shaking. Pour out the 0.5x SSC and replace it with 18 mL of fresh 0.5x SSC. Wash again for 10 min at 42 °C without shaking.

5. Visualization of the RNA Probe

NOTE: For visualizing the probe, BM purple will be used during the development process. However, before this step, several washes are required to prepare the samples for staining.

1. Wash the slides with 18 mL of alkaline phosphatase buffer (AP-buffer) without MgCl₂ for 1 min. Pour out the AP-Buffer without MgCl₂, and add 18 mL of 1x Boehringer-Mannheim blocking buffer diluted with maleic acid buffer. Incubate for at least 1 h at room temperature in a slide mailer with gentle shaking.
   NOTE: The Boehringer-Mannheim blocking buffer and maleic acid buffer used were premade and purchased in the DIG Wash and Block Buffer Set (available commercially).
   1. Alternatively, incubation overnight at 4 °C can be performed. A longer blocking period will lessen the appearance of non-specific binding.
2. Prepare the 20 mL of diluted DIG anti-digoxigenin-AP Fab fragments (anti-DIG antibody = 2 µL of anti-DIG antibody + 20 mL 1x Boehringer-Mannheim blocking buffer). This is enough for use in 1 plastic slide mailer. More of this solution must be prepared if more than one slide mailer is to be used.
3. Add the anti-DIG antibody to a new sterile slide mailer, and transfer the slides to the slide mailer with the anti-DIG antibody solution. Incubate at room temperature for 3 h with gentle shaking.
4. After incubation, pour out the anti-DIG antibody and wash the slides in the slide mailer with 18 mL of AP-Buffer without MgCl₂ for 5 min with gentle shaking.
5. Pour out the AP-Buffer without MgCl_2, and add 18 mL of AP-Buffer. Wash for 5 min with gentle shaking. After the 5 min incubation, pour off the AP-Buffer, replace it with 18 mL of fresh AP-Buffer, and wash for 5 min with gentle shaking.
6. In the dark, pour out the AP-Buffer and add 18 mL of BM purple to the slide mailer. Incubate the slides at room temperature in the dark, checking for purple color development every ½ h. Reaction times for visualization will vary based on the probe that is being developed.
   NOTE: Instead of BM purple visualization, development solution can be made using 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT). See Table 1 for instructions.
7. Stop color development by transferring the slides to a new sterile slide mailer with 18 mL of Tris-EDTA (TE) buffer for 5 min at room temperature, in the dark.
8. Pour off the TE buffer and add 18 mL of RNase-free water to the slide mailer and wash for 1 min, in the dark.
9. Remove the slides from the water, and dry them around the edges of the tissue. Add glycerol mounting medium and place the coverslips. Store the slides at 4 °C until pictures are ready to be taken.