TITLE: Hybridization Chain Reaction on paraffin sections 
AUTHORS: Astrid Deryckere, Ruth Styfhals, Ali Elagoz, Eve Seuntjens

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>This protocol has been optimized for HCR-IHC on paraffin sections of </span><span style = "font-style:italic;">Octopus vulgaris</span><span> embryos. More detailed information about our experiments can be found in the following publication: </span><a href="https://elifesciences.org/articles/69161" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://elifesciences.org/articles/69161</span></a></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>This protocol also led to successful results on killifish and mouse brain paraffin sections. The protocol is largely based on the recommended protocol by Molecular Instruments (</span><a href="https://www.molecularinstruments.com/hcr-rnafish-protocols" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.molecularinstruments.com/hcr-rnafish-protocols</span></a><span>) and on the protocol currently being used by the laboratory of Cris Niell (</span><a href="https://nielllab.uoregon.edu" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://nielllab.uoregon.edu/</span></a><span>).</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>For designing our HCR probes, we make use of the probe generator (</span><a href="https://github.com/rwnull/insitu_probe_generator" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/rwnull/insitu_probe_generator</span></a><span>;  </span><a href="https://www.biorxiv.org/content/10.1101/2021.04.22.439825v1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.biorxiv.org/content/10.1101/2021.04.22.439825v1</span></a><span>) and we order our probe sets from IDT (Integrated DNA Technologies).</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><br/><br/></div>

[STEPS]
?. [Tissue preparation]
Fix tissue overnight in 4 % paraformaldehyde (PFA) in phosphate buffered saline (PBS-DEPC) at 4°C.
?. [Tissue preparation]
Wash and store in PBS-DEPC at 4°C.
?. [Tissue preparation]
Transfer tissues to embedding cassettes (Tissue-Tek Biopsy - Chamber Cassette, Sakura). Immerse the cassettes in 0.9% NaCl in DEPC-water overnight, and then in 50% EtOH for a couple of hours (depending on tissue size) before progressive dehydration and paraffin-embedding using an ExcelsiorTM AS Tissue Processor and HistoStarTM Embedding Workstation (Thermo Scientific).
?. [Tissue preparation]
Make 6 μm-thick paraffin sections on superfrost plus slides and let dry overnight at 37°C. Slides can then be stored at room temperature.
?. [Day 1 (2.5 hours)]
Deparaffinization, Rehydration and permeabilization
?. [Day 2 (3 hours)]
PreparationPrepare wash solutions in coplin jars/falcons and pre-warm in the oven to 37°C before use.Cover with parafilm to avoid evaporation.
?. [Day 1 (2.5 hours)]
Bake slides (in a glass holder) at 65°C for 30 min in an oven.
?. [Day 1 (2.5 hours)]
Rehydrate the tissues by immersing the slides in a series of glass coplin jars under a chemical fume hood.4 minutes in Xylene 1 100%
4 minutes in Xylene 2 100%4 minutes in EtOH 100%4 minutes in EtOH 100%4 minutes in EtOH 100%
?. [Day 1 (2.5 hours)]
Move slides back to the oven and bake for 5 min at 65°C. Change the oven temperature to 37°C . Prepare the humidified chamber with kimwipes and autoclaved MQ.
?. [Day 1 (2.5 hours)]
Draw a line with a PAP pen at the white border of the slides.
?. [Day 1 (2.5 hours)]
Air dry for 5 minutes.
?. [Day 1 (2.5 hours)]
Add 800 μl of proteinase K solution per slide and make sure all sections are covered. Incubate slides in a humidified chamber for 5 min at 37°C. Move the hybridization buffer to ice.
?. [Day 1 (2.5 hours)]
Prepare the proteinase K solution: 1 μl of proteinase K in 3 ml of PBS-DEPC (Proteinase K rec PCR grade, art. 3115887001, Roche).
?. [Day 1 (2.5 hours)]
Wash 2 x 2 min in autoclaved MQ in a glass coplin jar.
?. [Day 1 (2.5 hours)]
Hybridization
?. [Day 1 (2.5 hours)]
Pre-warm oven and hybridization buffer to 37°C.
?. [Day 1 (2.5 hours)]
Remove as much of the solution as possible from the slide (pipet/kimwipe).
?. [Day 1 (2.5 hours)]
Add 200 ul of probe hybridization buffer on top of the tissue sample (contains formamide, dispose appropriately). Place a piece of parafilm on top to spread the volume across the slide. Wear a mask suitable for organic solvents or work under a chemical fume hood.
?. [Day 1 (2.5 hours)]
Pre-hybridize slides for 30 min inside the humidified chamber at 37°C.
?. [Day 1 (2.5 hours)]
Thaw probes on ice, spin down before using.
?. [Day 1 (2.5 hours)]
Prepare probe solution by adding 0.3 pmol of each probe mixture to 75 ul of probe hybridization buffer at 37°C (0.6 ul per probe).
?. [Day 1 (2.5 hours)]
Remove as much of the pre-hybridization solution as possible (with a pipet/kimwipe). Do this slide per slide. Make sure the paper within the humidified chamber is wet enough.
?. [Day 1 (2.5 hours)]
Add 75 ul of probe solution on top of the tissue sample.
?. [Day 1 (2.5 hours)]
Place a hybrislip (Grace Bio-labs) on the slide (this will cause the probe solution to distribute equally across all sections) and incubate overnight (12-16h) in the 37°C humidified chamber.
?. [Day 2 (3 hours)]
Wash steps
?. [Day 2 (3 hours)]
Remove the hybrislip without disturbing the tissue by immersing the slides in probe wash buffer. Wear a mask when dealing with the probe wash buffer, contains formamide!
?. [Day 2 (3 hours)]
Replace paper in the humidified chamber. Add autoclaved MQ to the chamber if necessary.
?. [Day 2 (3 hours)]
Removal of excess probes by incubating slides at 37°C in a glass coplin jar.
25% 5X SSCT (10 ml)/75% probe wash buffer (30 ml) for 15 min
50% 5X SSCT (20 ml)/50% probe wash buffer (20 ml) for 15 min
75% 5X SSCT (30 ml)/25% probe wash buffer (10 ml) for 15 min(Thaw hairpins on ice in the dark and move the amplification buffer to room T)100% 5x SSCT for 15 min (40 ml)100% 5x SSCT for 5 min at room T
?. [Day 2 (3 hours)]
Amplification stage
?. [Day 2 (3 hours)]
Take off SSCT solution as much as possible and replace PAP pen if needed.
?. [Day 2 (3 hours)]
Add 800 μl of amplification buffer on top of the tissue sample and pre-amplify in a humidified chamber for at least 30 min at room T.
?. [Day 2 (3 hours)]
Separately prepare 9 pmol (per slide) of H1 and H2 in separate PCR tubes. Specifically, pipet 1.5 μl of each hairpin [3 μM stock (4.5 pmol for H1 and 4.5 pmol for H2) in hairpin storage buffer] in a separate PCR tube.
?. [Day 2 (3 hours)]
In a PCR thermocycler: heat hairpins at 95 °C for 90s. Immediately put hairpins on ice for 5 minutes, and then leave hairpins at room temperature for 30 minutes IN THE DARK.
?. [Day 2 (3 hours)]
Prepare the hairpin solution by adding all snap-cooled hairpins to 75 ul of amplification buffer at room T. (Add equal amounts of amplification buffer to both hairpin 1 and 2 and then add hairpin 1 to hairpin 2)
?. [Day 2 (3 hours)]
Remove pre-amplification buffer as much as possible (one by one).
?. [Day 2 (3 hours)]
Add 75 ul of the hairpin solution on top of the tissue sample.
?. Place hybrislip on top and incubate overnight (12-16h) in a dark humidified chamber at room temperature.
?. [Day 3 (2 hours)]
Immerse slide in 5x SSCT at room T to release hybrislip.
?. [Day 3 (2 hours)]
Remove excess hairpins by incubating the slides in coplin jars with 5x SSCT: 3 x 10 min
?. [Day 3 (2 hours)]
Continue with IHC as normal if desired. To avoid loss of signal: try and minimize the incubation times and pay attention to the pH (PBS should have a pH of exactly 7.4).
?. [Day 3 (2 hours)]
Move slides to a coplin jar with SSCT-DAPI 1:1000. And incubate for 20 min in the dark.(MQ decreases the signal intensity).
?. [Day 3 (2 hours)]
Incubate 5 min in SSCT.
?. [Day 3 (2 hours)]
Add 100 ul of Mowiol to the slide and add a cover slip. Let the slides dry and store at 4°C protected from the light.