# **Hybridization Chain Reaction (HCR) In Situ Protocol** `Protocol Based on the [Patel Lab](http://www.patellab.net/) Hybridization Chain Reaction (HCR) In Situ Protocol available [here](https://www.protocols.io/view/hybridization-chain-reaction-hcr-in-situ-protocol-bunznvf6).` *This protocol largely follows the HCR v3.0 protocol for whole-mount Drosophila embryos (Choi et al. 2018). # Background In situ Hybridization chain reaction (HCR) is a modern molecular technique for visualizing gene expression in tissues. The technique utilizes short nucleotide probes that form stable hairpin structures (Metastable Small Conditional RNAs, scRNAs) that are able to polymerize only upon exposure to a target DNA initiator fragment. The introduction of the initiator triggers a cascade of hybridization events that yields a fluorescently labelled polymer that is bound to a sequence of interest. **Hybridization Chain Reaction (HCR) mechanism** ![](https://upload.wikimedia.org/wikipedia/commons/a/a4/Hybridization_Chain_Reaction_%28HCR%29_mechanism.png) Image source: https://commons.wikimedia.org/wiki/File:Hybridization_Chain_Reaction_(HCR)_mechanism.png HCR represents an improvement in both technical ease and specificity over previous generations of in situ hybridization protocols which largely relied on colorometric methods of labeling mRNA and were plaqued with high background. ## Probes We have had our probes synthesized by Molecular Instruments (Los Angeles, CA. USA). They recommend starting with a minimum of 1.4kb of sequence with 20 probe sets created from this. ## Preparation of solutions **10X PBS** *For 1 L of solution:* 2.56 g of NaH2PO4•H2O 11.94 g of Na2H2PO4 102.2 g of NaCl Fill up to 50 mL with distilled H2O *Please Note*: *You can prepare 1X PBS by diluting 1:10 with distilled H2O. Adjust pH to 7.40 with NaOH or HCl after dilution to 1X. Both 1X and 10X PBS can be kept indefinitely at room temperature.* **PTw** *For 50 mL of solution:* 5 mL of 10X PBS 500 µL of 10% Tween 20 Fill up to 50 mL with distilled H2O **20x SSC** *For 50 mL of solution:* 8.77 g of NaCl 4.41 g of sodium citrate Fill up to 50 mL with distilled H2O Adjust pH with 14 N HCl to 7.0 **5X SSCT** *For 40 mL of solution:* 10 mL of 20X SSC 400 µL of 10% Tween 20 Fill up to 40 mL with distilled H2O **Probe hybridization buffer (Store at -20°C)** *For 40 mL of solution:* 12 mL formamide 10 mL of 20× SSC 360 μL 1 M citric acid, pH 6.0 400 μL of 10% Tween 20 200 μL of 10 mg/mL heparin 800 μL of 50X Denhardt’s solution 8 mL of 50% dextran sulfate (high molecular weight) Fill up to 40 mL with distilled H2O **Probe wash buffer (Store at -20°C)** *For 40 mL of solution:* 12 mL formamide 10 mL of 20X SSC 360 μL 1 M citric acid, pH 6.0 400 μL of 10% Tween 20 200 μL of 10 mg/mL heparin Fill up to 40 mL with distilled H2O **Amplification Buffer (Store at 4°C)** *For 40mL of solution:* 10 mL of 20X SSC 400 μL of 10% Tween 20 8 mL of 50% dextran sulfate Fill up to 40 mL with distilled H2O **50% dextran sulfate ** *For 40 mL of solution:* 20 g of dextran sulfate powder (high molecular weight) Fill up to 40 mL with distilled H2O **Detergent Solution** *For 500 mL of Solution:* 50.0 mL 10% SDS (filtered) 12.5 mL 20% Tween 25.0 mL 1M Tris-HCl, pH 7.5 1.0 mL 0.5 M EDTA, pH 8.0 15.0 mL 5 M NaCl Fill up to 500 mL with distilled H2O **Glycerol Solutions** Prepare 50% and 70% glycerol solutions by mixing the appropriate volumes of ultrapure glycerol with 1X PBS (pH 7.40). Acidic glycerol will cause rapid loss of HCR probe signal. ## Protocol **Fixation:** Dissect tissue and fix for 10-40 minutes as you would for standard in situ or antibody staining. For fixation, we generally use 3.2% paraformaldehyde mixed in 1X artificial seawater (for Artemia and Parhyale) or 1X PBS (for Junonia, Antheraea, and Anthoscurria). For Drosophila and Tribolium, a standard heptane/PEM-FA fixation, with subsequent methanol devitillenization is followed (detailed protocol in Patel 1994). Samples may be dehydrated into 100% methanol after fixation and stored at -20°C for several years without a problem. This dehydration in methanol is not strictly necessary, but often makes the samples less prone to floating in later steps of the protocol. # **Day 1** **Step 1: Perform PTw Washes:** *Note: If your embryos/tissue are stored in methanol, first rehydrate into PTw. For larger embryos, we recommend rehydrating step-wise 75/50/25% methanol in PTw).* 1 × 10min PTw wash 1 × 5min PTw wash 1 × 5min PTw wash **Step 2: Permeabilize** in 300-500µL Detergent Solution for 30min at room temperature. *During this step, pre-warm probe hybridization buffer to 37°C (200µL/tube).* **Step 3: Pre-hybridize** embryos in pre-warmed 200µl of probe hybridization buffer for 30min at 37°C. *Note for reusing probe: Probe is presumably already in correct concentration, pre-warm to 37°C, then skip to Step 5. Number of reuses will depend on sample type and concentration used.* **Step 4: Prepare probe solution** by adding 0.8pmol (0.8µl of probe from 1uM stock solution) of each probe mixture to 200µL of probe hybridization buffer at 37°C. *Troubleshooting tip: If signals are weak, try increasing probe concentration up to 2-3x. This increased probe concentration can be useful when dealing with shorter than recommended starting sequences (fewer probe sets).* **Step 5: Remove the pre-hybridization solution** and add the probe solution. **Step 6: Incubate embryos overnight** (12-16h) at 37°C. *Troubleshooting tip: Extending incubation period up to 20 hours and/or increasing probe concentration up to 5-fold may be useful, especially with probes that have less than 20 probe pairs.* # **Day 2** **Step 7:** *Pre-heat probe wash buffer to 37°C before use, equilibrate amplification buffer to room temperature before use, and pre-set heat block to 95°C.* **Step 8:** Pre-heat probe wash buffer to 37°C before use, equilibrate amplification buffer to room temperature before use, and pre-set heat block to 95°C. **Step 9:** Wash samples 4 × 15min with 1mL of pre-warmed probe wash buffer at 37°C. 1 × 15min Probe Wash Buffer 1 × 15min Probe Wash Buffer 1 × 15min Probe Wash Buffer 1 × 15min Probe Wash Buffer **Step 10:** Wash samples 2 × 5min with 1mL of 5X SSCT at room temperature. 1 × 5min 5x SSCT 1 × 5min 5x SSCT **Step 11:** Pre-amplify embryos with 1mL of pre-equilibrated amplification buffer for 30min at room temperature. *Make sure to pre-equilibrate amplification buffer to room temperature before use.* 1 × 30min Amplification Buffer **Step 12:** During the pre-amplification step, prepare hairpins. Mix 2µL (3µM stock) of each hairpin h1 and 2µL of each hairpin h2 in 100µL of amplification buffer at 95°C for 90sec, then cool to room temperature in a dark drawer for 30min. *Troubleshooting tip: Doubling hairpin concentration can help to boost signal.* ***Note: If reusing hairpins from prior experiment, heat and cool, then skip to Step 13.*** **Step 13:** Remove the pre-amplification solution and add the hairpin solution. **Step 14:** Incubate the embryos overnight (2–16h) in the dark at room temperature. # **Day 3** **Step 15:** **SAVE HAIRPIN MIXTURE.** Can be reused multiple times. Save used hairpin mixtures at -20°C. Remove excess hairpins by washing with 1mL of 5X SSCT at room temperature: 1 × 5min 5X SSCT 1 × 5min 5X SSCT 1 × 30min 5X SSCT 1 × 30min 5X SSCT 1 × 5min 5X SSCT **Step 16:** Incubate embryos in 50% glycerol solution (in 1X PBS) with DAPI (skip if DAPI is not needed): 30min-1h if using 1.0µg/mL DAPI 2 h or overnight at 4°C if using 0.1µg/mL DAPI **Step 17:** Replace DAPI glycerol with 50%-70% glycerol (in 1X PBS) and store at 4°C. *Troubleshooting tip: 1X PBS must be at pH 7.40, deviation from this may result in rapid loss of signal.* **Step 18:** Mount and image embryos.