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Method Article

Wholemount In Situ Hybridization for Astyanax Embryos

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DOI:

10.3791/59114

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March 2nd, 2019

 ,  ,  , 
1Department of Biological Sciences, University of Cincinnati

In This Article

Summary

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

Abstract

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Introduction

In situ hybridization is a common method for staining fixed tissues to visualize gene expression patterns1. This technique has been performed for years in other traditional2 and non-traditional3 model systems, for a variety of biological studies. However, several steps and reagents are necessary to successfully perform this procedure. For investigators who have never performed this technique, initiating the process can be intimidating owing to the many steps involved. Further, the lengthy nature of this procedure lends itself to technical errors, which can be challenging to troubleshoot.

The overall goal of this article is to present a simple and straightforward method that will render this hybridization technique accessible to a wide audience. To reduce the introduction of errors, we present a straightforward approach that yields high quality gene expression staining and minimizes non-specific background signal. This procedure is similar to other approaches developed in traditional model systems, such as Danio rerio4. Here, we aim to facilitate careful implementation of each step using a downloadable checklist (Supplemental File 1), to promote careful implementation of the protocol. The rationale for doing this is to facilitate organization through the many steps involved in this procedure. This article is appropriate for researchers interested in performing whole-mount in situ hybridization in developing embryos, but have not yet performed the procedure. The advantage of the chosen approach for Astyanax researchers is that it has been tested and proven in both cavefish and surface fish morphs, thereby facilitating comparative expression analyses. The presented method can be used by researchers in studies on Astyanax and other systems.

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Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Cincinnati (Protocol #10-01-21-01).

1. Fixation

  1. Isolate desired number of Astyanax mexicanus embryos from a breeding tank and fix ~50 embryos at a time. If embryos are large and old, it may be necessary to fix 25 at a time to ensure even fixation.
  2. Depending on the age of the embryo, utilize the IACUC-approved method of anesthesia. For older embryos with a functioning nervous system, sacrifice embryos via anesthetic overdose. Accordingly, place embryos in a solution of ~1% tricaine (buffered to pH 7.4) to minimize pain and discomfort for the organism.
  3. Once the embryos are unresponsive to touch, replace system water containing tricaine, and add ~1 mL of 1x phosphate-buffered saline (PBS, pH 7.4).
  4. Remove the PBS solution, and add 1 mL of 4% paraformaldehyde (PFA). Fix embryos overnight at 4 °C.
    CAUTION: PFA is hazardous (i.e., it is flammable and is a skin and lung irritant), handle with care.

2. Dehydration

  1. To dehydrate the embryos, remove the fixative solution and rinse with 1 mL of PBS. Place embryo-containing vials at an angle (between 30° and 45°), on a platform shaker during rinse. Continue to wash the embryos twice, 5 min per wash.
  2. If embryos still have a chorion, place all 50 embryos into a 100 mm x 25 mm Petri dish, and carefully isolate them from the chorions using two sets of Watchmaker’s forceps (e.g., #5 forceps) under a microscope.
    NOTE: During the steps described below, and for the remainder of the protocol, carefully remove all liquid from the previous step using clean, glass Pasteur pipettes before adding the next solution
  3. Dehydrate the embryos in a series of increasingly concentrated washes of methanol (MeOH) described below. The dilutions are based on a 1 mL total volume with 500 µL of solution going into each 4 mL glass vial. Perform all dehydration steps at room temperature (RT) on platform shaker.
    1. Carefully remove the PBS solution. Add a 25% MeOH solution (250 µL of MeOH + 750 µL of PBS). Gently shake on a platform shaker for 5 min.
    2. Carefully remove the 25% MeOH solution. Add a 50% MeOH solution (500 µL of MeOH + 500 µL of PBS). Gently shake on a platform shaker for 5 min.
    3. Carefully remove the 50% MeOH solution. Add a 75% MeOH solution (750 µL of MeOH + 250 µL of PBS). Gently shake on a platform shaker for 5 minutes.
    4. Carefully remove the 75% MeOH solution. Add a 100% MeOH solution (1 mL of MeOH). Gently shake on a platform shaker for 5 min. Repeat this step 3 times.
  4. At this point, store dehydrated embryos, as needed, in their glass vials at -20 °C (long term). Alternatively, proceed directly to day 1 of the protocol.

3. Day 1: Rehydration

  1. Obtain dehydrated embryos from -20 °C freezer (or proceed directly from step 2.5).
  2. Sort the embryos using a Pasteur pipette. One can sort based on morphotype (i.e., cave and/or surface), and the number of genes assessed in each experiment. There are usually no more than 12 embryos per vial once sorted. To maintain organization, use colored lab tape to designate vials and pipettes for each gene. Embryos will stay in the same vial throughout the entire protocol.
    NOTE: Place the tip of the Pasteur pipette in 100% EtOH to sterilize it between uses.
  3. Set a shaking water bath to 70 °C to be used in a later step. Carefully, draw out the MeOH in vials of sorted embryos and replace with 500 µL of new 100% MeOH. Wash briefly (~1 min) on platform shaker.
  4. Rehydrate embryos in an increasing concentration of 1x PBS with Tween 20 (PBT, see below) on platform shaker. The dilutions are based on a 1 mL final dilution volume with 500 µL going into each vial.
    1. Add a 25% PBT solution (250 µL of PBT, 750 µL of MeOH). Gently shake on a platform shaker for 5 min.
    2. Carefully remove the 25% PBT solution. Add a 50% PBT solution (500 µL of PBT, 500 µL of MeOH). Gently shake on a platform shaker for 5 min.
    3. Carefully remove the 50% PBT solution. Add a 75% PBT solution (750 µL of PBT, 250 µL of MeOH). Gently shake on a platform shaker for 5 min.
    4. Carefully remove the 75% PBT solution. Add a 100% PBT solution (1 mL of PBT). Gently shake on a platform shaker for 5 min. Repeat this step 3 times.

4. Day 1: Digestion and fixation

  1. Prepare a proteinase K (PK) solution by adding 1 µL of PK (20 mg/mL) to 2 mL of PBT.
  2. In anticipation of subsequent steps, obtain frozen aliquots of hybridization buffers (Hyb- and Hyb+; see Supplemental File 2 and Supplemental File 3) and PFA from -20 °C storage.
    1. Allow PFA to thaw at RT.
    2. Place aliquots of Hyb- and Hyb+ in a rotating 70 °C water bath. Place all reagents and vials inside a small “gasket” with a mesh bottom, inside the floating water bath apparatus. This enables simple addition and removal of tubes and vials from the rotating 70 °C water bath.
  3. Gently add PK solution to the vial(s) of embryos ensuring all tissues are completely covered with solution. Digest embryos for ~12 min in PK working solution on the platform shaker.
    NOTE: The length of digestion can be varied by the investigator to ensure optimal results.
  4. Gently draw off the PK solution, and briefly flood the vial with PBT to dilute any remaining PK.
  5. Draw off the PBT solution and replace with 500 µL of new PBT. Allow the solution to rinse on the platform shaker for 5 min.
  6. Draw off PBT and replace with 500 µL of thawed 4% PFA. Allow the embryos to incubate for 20 min on the platform shaker at RT.
  7. Draw off the 4% PFA, and briefly flood the vial with PBT to dilute any remaining PFA. Draw off the PBT, and replace with 500 µL of fresh PBT. Allow embryos to rinse for 5 min on the platform shaker. Repeat this step 4 more times.

5. Day 1: Prehybridization

  1. Place 500 µL of pre-warmed Hyb- solution into the vial. Carefully place the vial in the 70 °C water bath (inside gaskets) without shaking, for 5 min.
  2. Draw off the Hyb- solution and flood the vial with 500 µL of pre-warmed Hyb+ solution. Place the vial back into the 70 °C water bath with shaking (40 rpm). Incubate for either 4 h, or overnight.
    NOTE: A 4 h incubation will yield a complete in situ protocol that will last for 4 days in total. Here, this step is presented as an overnight incubation, which will yield a protocol lasting 5 days in total.

6. Day 2: Hybridization

  1. Place an aliquot of Hyb+ from the -20 °C freezer into the shaking hot water bath for 5 min.
  2. Draw off the Hyb+ from the vial and replace with 500 µL of pre-warmed Hyb+. To this solution, carefully add 2 µL of RNA probe to each vial. Gently swirl the vial to ensure even distribution of the probe.
  3. Incubate the Hyb+ (with added probe) solution in the 70 °C hot water bath overnight while shaking at 40 rpm.
    NOTE: One can re-use Hyb+ (with probe) solution. For this, take Hyb+ with probe from the first run from the -20 °C freezer and place it in a hot water bath for 5 min. Replace Hyb+ from the day 1 protocol with Hyb+ with probe and allow incubating overnight in hot water bath.

7. Day 3: Solution preparation

  1. Prepare microcentrifuge tubes labeled Hyb+ with the “gene-of-interest” RNA probe. Prepare the series of dilutions that will be used during day 3.
    1. Using 6 separate tubes, prepare the following series of dilutions of Hyb- and saline sodium citrate (SSC, 0 to 100%) in a 1 mL volume, and place them in the 70 °C shaking water bath: Tube 1 = 100% Hyb- (1 mL of Hyb-): Tube 2 = 25% 2x SSC (250 µL of 2x SSC, 750 µL of Hyb-); Tube 3 = 50% 2x SSC (500 µL of 2x SSC, 500 µL of Hyb-); Tube 4 = 75% 2x SSC (750 µL of 2x SSC, 250 µL of Hyb-); Tube 5 = 100% 2x SSC (1 mL of 2x SSC); Tube 6 = 100% 0.2x SSC (2 mL of 0.2x SSC).
      NOTE: Be vigilant of the concentration of SSC, as it changes from 2x to 0.2x.
    2. Using 4 separate tubes, prepare the following series of dilutions of PBT and SSC in a 1 mL volume, and place at RT: Tube 1 = 25% PBT (250 µL of PBT, 750 µL of 0.2x SSC); Tube 2 = 50% PBT (500 µL of PBT, 500 µL of 0.2x SSC); Tube 3 = 75% PBT (750 µL of PBT, 250 µL of 0.2x SSC); Tube 4 = 100% PBT (1mL of PBT).
    3. Prepare a tube with 2 mL of maleic acid buffer containing Tween 20 (MABT) working solution.
    4. Prepare two 15 mL conical tubes of blocking solution. In each tube, add 0.2 g of blocking reagent to 10 mL of MABT (see Supplemental File 4). Place both tubes on a nutating mixer (or platform shaker) until completely dissolved in solution (up to 3 h).

8. Day 3: Probe removal

  1. Draw off Hyb+ (with probe) solution with a glass Pasteur pipette and place it into a sterile, labeled microcentrifuge tube. Retain this tube in the -20 °C freezer for future use (if probe-labeling is successful).
  2. Carefully add 500 µL of the warm SSC/Hyb- dilutions (indicated below). Incubate in each of the following solutions for 10 min each in the 70 °C shaking water bath.
    1. Incubate sequentially with 100% Hyb- (1 mL of Hyb-), 25% 2x SSC (250 µL of 2x SSC, 750 µL of Hyb-), 50% 2x SSC (500 µL of 2x SSC, 500 µL of Hyb-), 75% 2x SSC (750 µL of 2x SSC, 250 µL of Hyb-), 100% 2x SSC (1 mL of 2x SSC), 100% 0.2x SSC (2 mL of 0.2x SSC).
  3. Following the last step, incubate in each of the following solutions for 10 min each. All of the following incubations take place at RT on the platform shaker: 25% PBT (250 µL of PBT, 750 µL of 0.2x SSC), 50% PBT (500 µL of PBT, 500 µL of 0.2x SSC), 75% PBT (750 µL of PBT, 250 µL of 0.2x SSC), 100% PBT (1 mL of PBT).
  4. After a 10 min incubation, remove the 100% PBT, and add 500 µL of MABT into each vial. Repeat this step twice for 5 min.

9. Day 3: Blocking

  1. Remove MABT from each vial and flood with premixed blocking solution from one of the tubes (prepared in step 7.1.4). Place vial on a nutating mixer for ~4 h at RT.
  2. Add 2 µL of Anti-DIG-AP Fab fragments to the second vial of 10 mL blocking solution (prepared in step 7.1.4) and briefly vortex.
  3. Fill each vial almost completely with blocking solution (~5 mL) and place on nutating mixer overnight in a refrigerator at 4 °C.

10. Day 4: MABT Rinses

  1. Prepare a stock vial of 10% normal goat serum (NGS) in MABT (add 100 µL of NGS to 900 µL of MABT).
  2. Draw off the blocking solution in each vial and add 500 µL of NGS/MABT mixture into each vial. Allow the embryos to incubate for 25 min at RT on the platform shaker.
  3. Replace the NGS/MABT mixture with 500 µL of 100% MABT. Incubate for 30 min at RT on the platform shaker. Perform this rinse 11 more times throughout the day every 30 min.
  4. Fill the vial with 100% MABT, and place on a nutating mixer overnight in a refrigerator or walk-in chamber at 4 °C.

11. Day 5: Probe visualization

  1. Prepare a 50 mL aliquot of alkaline phosphatase (AP) buffer (see Supplemental File 5). Combine the following in a 50 mL conical tube wrapped in aluminum foil to limit light exposure: 5 mL of 1 M Tris (pH 9.5), 5 mL of 50 mM MgCl2, 5 mL of 1% Tween 20, 5 mL of 1 M NaCl, 30 mL of ddH2O.
  2. Remove MABT and replace with 1 mL of AP buffer (tube wrapped in foil). Let it wash for 5 min. Do this twice to ensure complete removal of MABT.
  3. Remove AP buffer and replace with 1 mL of AP buffer with 3.5 μL 5-bromo-4-chloro-3'-indolyphosphate (BCIP) and 4.5 μL of nitro-blue tetrazolium (NBT). Replace with freshly prepared AP buffer/NBT/BCIP once every hour until reaction is complete. Monitor closely, checking every 15 min, to allow the coloration reaction to take place until the desired level of staining has been achieved. If precipitate begins to form, replace the solution sooner.
  4. Stop the coloration reaction by rinsing the embryos in fresh 100% AP buffer (without NBT/BCIP) for 5 min. Continue rinses in PBT until optimal levels of signal (with minimal amounts of background staining) are achieved. Continue to rinse specimens with increasing dilutions of PBT in AP Buffer as follows: 25% PBT (250 μL of PBT, 750 μL of AP Buffer), rinse for 5 min; 50% PBT (500 μL of PBT, 500 μL of AP Buffer), rinse for 5 min; 75% PBT (750 μL of PBT, 250 μL of AP buffer), rinse for 5 min.
  5. Rinse embryos in ~5 mL of 100% PBT on nutating mixer until desired minimum background staining is reached. Switch out with fresh PBT several times. This could take up to several days.
  6. When rinsing is complete, wash embryos in 500 μL of sterile PBS on a platform shaker. Perform this rinse twice for 5 min. After PBS washes, post-fix the specimens in 500 μL of 4% PFA for 1 h at RT on a platform shaker. Alternatively, fix overnight in 1 mL of 4% PFA in the refrigerator at 4 °C.
  7. Replace the fixative with fresh, sterile PBS. Perform this rinse at least twice for 5 min. Place the embryos in ~4 mL of 100% sterile PBS, and store long term at 4 °C.

12. Imaging

  1. Make up an imaging plate in a Petri dish using 3% agarose and TAE buffer.
    NOTE: Quantities depend on how many plates are needed. Plates can be reused several times. It is recommended that a shallow rectangular mold is placed in the Petri dish while the gel is cooling in order to create a depression for containing the embryos on the plate.
  2. Place the embryos on the plate in the PBS.
    NOTE: It is best to gently pour embryos onto the plate instead of pipetting them out because it has been found that they will stick to the inside of plastic pipettes.
  3. Use light microscopy in order to visualize each embryo. Use a blunt probe to maneuver embryos to desired position.
  4. Take an image when the embryo is in desired position. Note that it is important to take images of embryos within a couple weeks of completed staining to avoid potential degradation of stain.

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Results

In this report, we provide a simple and straightforward approach to perform labeling of embryonic Astyanax specimens for high-quality gene expression analysis. This technique can be carried out in either four or five days, and each principal step in the procedure is represented in a color-coded flowchart (Figure 1). Once completed, stained embryos should harbor a dark purple chromatic label in tissues expressing the particular gene of interest. We ha...

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Discussion

Owing to the vulnerability of RNA to degradation, one of the most critical steps in the protocol concerns the sterile synthesis of the RNA probe. However, if a probe is carefully generated, and provides good results, it can be reused in subsequent staining reactions. A second crucial step is the careful production of all reagents used throughout the protocol. Since this protocol involves several days and many small steps, it is essential that all reagents are accurately produced, and stored in a sterile manner. Further, ...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors wish to thank members of the Gross lab for helpful comments on this manuscript. We wish to acknowledge four high school students who utilized this protocol during summer internships in 2017 and 2018, including Christine Cao, Michael Warden, Aki Li, and David Nwankwo. HL was supported by a UC Biology STEM Fellowship during the summer of 2017. This work was supported by grants from the National Science Foundation (DEB-1457630 to JBG), and the National Institutes of Dental and Craniofacial Research (NIH; DE025033 to JBG).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
10 mL Serological PipetteVWR89130-888
1000 mL Filtration UnitVWR89220-698
15 mL ConicalVWR-Greiner82050-278
25 mL Serological PipetteVWR89130-890
250 mL Filtration UnitVWR89220-694
5 mL Serological PipetteVWR89130-886
50 mL ConicalVWR-Falcon21008-940
500 mL Filtration UnitVWR89220-696
Anti-Digoxigenin-AP, Fab fragmentsSigma-Roche11093274910
BCIPSigma-AldrichB8503-1G1 g
Blocking SolutionSigma-Roche11 096 176 00150 g
Citric AcidFisher ScientificA104-500500 g
DIG RNA Labeling Kit (SP6/T7)Sigma-Roche11175025910
Eppendorf TubesVWR20170-577
EthanolFisher-Decon04-355-2231 Gal
FormamideThermo Fisher Scientific17899100 mL
Glass dram vialsVWR66011-0411 Dr
Glass PipettesFisher Scientific13-678-8A
HClThermal-ScientificPharmco-AAPER284000ACS500 mL
HeparinSigmaH3393-25KU
Magnesium Chloride-crystallineFisher ScientificM33-500500 g
Maleic AcidSigmaM0375-100g100g
MethanolFisher ScientificA452-44L
Molecular-grade Water (RNase-free)VWR7732-18-5500 mL
NaClFisher ScientificS271-33 kg
NaOH pelletsFisher ScientificS318-500500 g
NBT Substrate powderThermoFisher Scientific340351 g
Normal Goat SerumFisher-Invitrogen31873
Nutating MixerVWR82007-202
ParaformaldehydeSigma158127-500g500 g
PBS 10xFisher ScientificBP399-2020L
Proteinase K (200mg/10ml)Qiagen1913310 mL
Plastic PipettesVWR-Samco14670-147
RNAseSigmaR2020-250mL250 mL
Shaking Water Bath 12 LVWR10128-12612 L
Standard Analog ShakerVWR89032-092
TrisSigma Millipore-OmniPur9210-500GM500 g
tRNA YeastFisher-Invitrogen1540101125 mg
Tween 20SigmaP9416-50mL50 mL
Vortex-Genie 2Fisher Scientific-Scientific Industries, Inc50-728-002
Lithium Chloride (LiCl)Sigma-Aldrich203637-10G10 g

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Tags

Gene Expression AnalysisProteinase K DigestionHybridization BufferBlocking SolutionAP Buffer StainingLight Microscopy ImagingCavefish Surface FishRNA Probe Detection
Wholemount In Situ Hybridization for Astyanax Embryos
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