Oct 18, 2022

Public workspaceSHInE - Simultaneous HCR, Immunohistochemistry, Nuclear staining and EdU V.1

Integrative and Comparative Biology
DOI
dx.doi.org/10.17504/protocols.io.5qpvobnyzl4o/v1
  • 1Max Perutz Labs, University of Vienna, Vienna BioCenter, Dr. Bohr-Gasse 9/4, 1030 Vienna, Austria;
  • 2Research Platform “Rhythms of Life”, University of Vienna, Vienna BioCenter, Dr. Bohr-Gasse 9/4, A-1030 Vienna, Austria;
  • 3Research Platform "Single-Cell Genomics of Stem Cells", University of Vienna, Vienna BioCenter, Dr. Bohr-Gasse 9/4, A-1030 Vienna, Austria;
  • 4Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany;
  • 5Carl-von-Ossietzky University, Carl-von-Ossietzky-Straße 9-11 ,26111 Oldenburg, Germany
Alexander Stockinger
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DOI: dx.doi.org/10.17504/protocols.io.5qpvobnyzl4o/v1
Protocol Citation: Aida Coric, Alexander W. Stockinger, Petra Schaffer, Dunja Rokvic, Kristin Tessmar-Raible, Florian Raible 2022. SHInE - Simultaneous HCR, Immunohistochemistry, Nuclear staining and EdU. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvobnyzl4o/v1Version created by Alexander Stockinger
Manuscript citation:
Aida Ćorić, Alexander W Stockinger, Petra Schaffer, Dunja Rokvić, Kristin Tessmar-Raible, Florian Raible, A Fast And Versatile Method for Simultaneous HCR, Immunohistochemistry And Edu Labeling (SHInE),Integrative and Comparative Biology, 2023;, icad007,https://doi.org/10.1093/icb/icad007
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 10, 2022
Last Modified: October 18, 2022
Protocol Integer ID: 71099
Keywords: HCR, immunohistochemistry, in situ hybridisation, FISH, whole-mount, multiplexed, EdU, proliferation, Platynereis, Annelid
Abstract
This protocol allows the multiplexed use of four different molecular labelling techniques in whole-mount Platynereis tissues. In short, gene expression (via in situ HCR 3.0), cell proliferation (via EdU labelling), proteins (via Immunohistochemistry) and nuclei (DAPI/Hoechst staining) can be assessed in whole-mount tissue samples, allowing the co-visualisation of various types of molecules in the same specimen.

This protocol combines knowledge from multiple sources (see below), and is being submitted in parallel with a manuscript detailing the applications of the method. (We will add the reference as soon as it's available.)

We successfully applied this protocol to heads and posterior regenerates of the bristleworm Platynereis dumerilii, at various developmental stages of the animal. Given the general nature of the detected molecules, and the wide-spread use of the individual detection techniques, we anticipate that this protocol will be well applicable to a wider range of model systems.

References:
1) Choi HMT, Calvert CR, Husain N, Huss D, Barsi JC, Deverman BE, Hunter RC, Kato M, Lee SM, Abelin ACT, Rosenthal AZ, Akbari OS, Li Y, Hay BA, Sternberg PW, Patterson PH, Davidson EH, Mazmanian SK, Prober DA, Rijn M van de, Leadbetter JR, Newman DK, Readhead C, Bronner ME, Wold B, Lansford R, Sauka-Spengler T, Fraser SE, Pierce NA. 2016. Mapping a multiplexed zoo of mRNA expression. Development 143:3632–3637. doi:10.1242/dev.140137

2) Choi HMT, Schwarzkopf M, Fornace ME, Acharya A, Artavanis G, Stegmaier J, Cunha A, Pierce NA. 2018. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145:dev165753. doi:10.1242/dev.165753

3) Kuehn E, Clausen DS, Null RW, Metzger BM, Willis AD, Özpolat BD. 2021. Segment number threshold determines juvenile onset of germline cluster proliferation in Platynereis dumerilii. Biorxiv 2021.04.22.439825. doi:10.1101/2021.04.22.439825

4) Pende M, Vadiwala K, Schmidbaur H, Stockinger AW, Murawala P, Saghafi S, Dekens MPS, Becker K, Revilla-i-Domingo R, Papadopoulos S-C, Zurl M, Pasierbek P, Simakov O, Tanaka EM, Raible F, Dodt H-U. 2020. A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity. Sci Adv 6:eaba0365. doi:10.1126/sciadv.aba0365

5) Salic A, Mitchison TJ. 2008. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc National Acad Sci 105:2415–2420. doi:10.1073/pnas.0712168105

6) Tessmar-Raible K, Steinmetz PRH, Snyman H, Hassel M, Arendt D. 2005. Fluorescent two-color whole mount in situ hybridization in Platynereis dumerilii (Polychaeta, Annelida), an emerging marine molecular model for evolution and development. Biotechniques 39:460–464. doi:10.2144/000112023
Guidelines
  • Please use aliquots of common stocks, including all buffers, to avoid repeated freeze-thaw cycles.
  • The protocol is based on the detection of RNA, so minimize RNA degradation. It does not seem to be as critical as with sequencing experiments, but keep samples on ice whenever indicated, and work quickly in a clean setting.
  • Protect samples from light once fluorescent dyes have been added to them!
  • HCR signal seems to be best in the first 5 days after preparation of the samples, so ideally, image your samples right after preparing them.
  • Many of the steps in this protocol can be optimized for individual samples or experimental conditions. We tried to indicate these steps. If you do come up with improved conditions for certain samples, we want to encourage you to upload a modified version of this protocol!

For multi-color fluorescence labeling, check whether the microscope you intend to use has lasers and filters available. HCR signal in general seems to only be visible on the confocal microscope. You can also check for bleed through risk and plan accordingly, for example using this tool:
https://www.thermofisher.com/order/fluorescence-spectraviewer

Probe maker:
Probes can be ordered from Molecular Instruments, alongside the amplifier hairpins that contain the fluorophore.
To design HCR probes yourself, you can use a Python based tool developed by Ryan Null in the Özpolat lab at WUSTL. It can be found here:
https://github.com/rwnull/insitu_probe_generator
and was first referenced in
Kuehn E, Clausen DS, Null RW, Metzger BM, Willis AD, Özpolat BD. 2022. Segment number threshold determines juvenile onset of germline cluster expansion in Platynereis dumerilii.J Exp Zoology Part B Mol Dev Evol338:225–240. doi:10.1002/jez.b.23100

In short, use a target sequence and the probe generator will generate sets of probes that contain the initiator sequence of your choice.

* Probes can either be acquired through https://www.molecularinstruments.com, or self-designed (we use this python tool https://github.com/rwnull/insitu_probe_generator) and then ordered as oligos, for example through https://www.idtdna.com/pages/products/custom-dna-rna/dna-oligos/custom-dna-oligos/opools-oligo-pools

For more detailed instructions on EdU pulse-and-chase experiments in Platynereis dumerilii, please refer to
Zattara, E. E. & Özpolat, B. D. Developmental Biology of the Sea Urchin and Other Marine Invertebrates.Methods Mol Biology2219, 163–180 (2020).


Materials
Buffer recipes

Hybridization buffer (toxic, store at -20° C):
1x concentrationFor 40ml
30% formamide12 mL formamide
5x sodium chloride sodium citrate (SSC)10 mL of 20× SSC
9 mM citric acid (pH 6.0)360 μL 1 M citric acid, pH 6.0 
0.1% Tween 20400 μL of 10% Tween 20 
50 μg/mL heparin200 μL of 10 mg/mL heparin
1x Denhardt’s solution800 μL of 50× Denhardt’s solution 
10% dextran sulfate8 mL of 50% dextran sulfate
 Fill to 40 ml with ultrapure H2O

Wash buffer (toxic, store at -20° C):

1x concentrationFor 40ml
30% formamide12 mL formamide
5× sodium chloride sodium citrate (SSC)10 mL of 20× SSC
9 mM citric acid (pH 6.0)360 μL 1 M citric acid, pH 6.0 
0.1% Tween 20400 μL of 10% Tween 20 
50 μg/mL heparin200 μL of 10 mg/mL heparin
 Fill up to 40 ml with ultrapure H2O

Amplification buffer (store at 4° C):

1x concentrationFor 40ml
5× sodium chloride sodium citrate (SSC)10 mL of 20× SSC
0.1% Tween 20400 µl of 10% Tween 20
10% dextran sulfate8 mL of 50% dextran sulfate
 Fill up to 40 ml with ultrapure H2O

5x SSCT (store at 4° C):

1x concentrationFor 40ml
5× sodium chloride sodium citrate (SSC)10 mL of 20× SSC
0,1% Tween 20400µl of 10% Tween 20
 Fill up to 40µl with ultrapure H2O


1X PTW:
1X PBS with 0.1% Tween-20

Tissue Clearing:
1.Pende, M.et al. A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity.Sci Adv6, eaba0365 (2020).


Reagents
ABC
ReagentManufacturerProduct Number
Citric acid monohydrateSigma-AldrichC1909
Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 488 dyeInvitrogen™ C10337
Denhardt's solution 50xInvitrogen™750018
Dextran Sulfate 50% solutionMerck MilliporeS4030 
FormamideSigma-Aldrich47671
GlycineRoth3908.3
HeparinSigma-AldrichH3393
Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water Invitrogen™H3570
Magnesiumchlorid hexahydrate (MgCl₂)VWR25.108.295
Methanol VWR20847
Methanol, suitable for HPLCSigma-Aldrich34860
Paraformaldehyde (PFA)Sigma-Aldrich441244
Phosphate buffered saline (PBS)Sigma-AldrichP4417
Proteinase KSigma-Aldrich1.245.680.100
Saline-Sodium Citrate buffer 20× Concentrate (SSC 20X) Sigma-AldrichS6639
SlowFade™ Diamond Antifade MountantMolecular Probes™ S36972
ThymidineSigma-AldrichT9250
Tween 20Sigma-AldrichP1379


Optional: EdU incubation
Optional: EdU incubation
Incubate worms with Concentration10 micromolar (µM) EdU in artificial sea water (ASW) (EdU aliquots in both DMSO and H2O work fine) for your desired pulse length.

Optional
Toxic
Optional: Chase out EdU with Concentration3 millimolar (mM) thymidine for Duration01:00:00 ; then replace thymidine solution with fresh ASW and incubate for desired chase length

1h
Optional
Day 1: Fixation, Dehydration
Day 1: Fixation, Dehydration
Dissect tissue of interest
Anesthetize worms in a 50% mix of 7.5% (w/v) MgCl2 and ASW
Dissect the tissue of interest; transfer it to a 1,5ml microcentrifuge tube pre-filled with 1ml ASW TemperatureOn ice

Note: the protocol is also compatible with whole animals, but will require larger volumes and more reagent accordingly.
Fix the samples
Replace the ASW in the tube with 1ml 4%PFA/1XPTW (carefully pipet away the ASW; samples should sink to the tube bottom). Fix samples for Duration01:00:00 at TemperatureRoom temperature shaking gently on a rocking platform

Make sure the samples are swimming back and forth freely in the tube; dislodge stuck samples by gently flicking the tube.
1h
Toxic
Dehydrate and store samples
Dehydrate the samples through a series of increasing MeOH concentrations diluted in 1X PTW.
Perform these steps on ice, and wait until the samples have settled down at the bottom of the well after each increase in MeOH concentration (takes around 1-3 minutes)

  • 25% MeOH in PTW Duration00:03:00
  • 50% MeOH in PTW Duration00:03:00
  • 75% MeOH in PTWDuration00:03:00
  • Wash in100% MeOH Duration00:01:00
  • Wash in100% MeOH Duration00:01:00
11m
Toxic
Store samples at -20° C for at least DurationOvernight . Longer storage is possible (several months in our hands, but even longer storage might be possible).

Pause
Overnight
Day 2: Rehydration, digestion, probe hybridization
Day 2: Rehydration, digestion, probe hybridization
1h 45m
1h 45m
Re-hydrate samples through a series of decreasing MeOH concentrations in 1X PTW on ice.
  • 75% MeOH in PTW Duration00:03:00
  • 50% MeOH in PTWDuration00:03:00
  • 25% MeOH in PTWDuration00:03:00
  • Wash 2x in 1X PTWDuration00:03:00



12m
Toxic
Perform proteinase K treatment with 1ml of Proteinase K in PTW according to table below, at TemperatureRoom temperature .
Critical
This is a critical, time-sensitive step and all reagents needed for washes after digestion should be prepared in advance. Concentrations and digestion times should be adjusted to individual tissues and possibly differences in proteinase K batches.
TissuePK concentrationtime
Heads, Blastemas, adult tissues100µg/mL5'
Larvae, <1dpf100µg/mL30 seconds
Larvae, 3dpf100µg/mL2'

Critical
Briefly rinse 2x with 1 ml of glycine wash buffer to stop digest. Work TemperatureOn ice from here on.
Wash once for Duration00:05:00 with 1x PTW.


5m
Post-fix in 1mL 4%PFA diluted in 1xPTW for Duration00:20:00 shaking while still TemperatureOn ice .
20m
Toxic
Wash samples 2x Duration00:05:00 in 1ml PTW.

5m
Optional: tissue clearing
If clearing with DEEP-CLEAR is wanted, perform it at this step. Worm tissue can be cleared at Temperature37 °C for Duration00:15:00 at 300 rpm on a heat block. Clearing time should be optimised for different tissues. Wash the samples in PTW thoroughly after clearing (3x Duration00:05:00 ).

Note: in Platynereis tissues, the effects of clearing depend on the tissue sampled. Opaque tissues such as eyes benefit from clearing, while naturally transparent tissues like blastemas don't.

For details on tissue clearing, please refer to
Pende, M.et al. A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity.Sci Adv6, eaba0365 (2020)
20m
Optional
Probe hybridization with HCR probe(s) of choice.

Note: a weak probe signal might be improved by increasing the probe concentration; several labs have reported successfully reusing the probe solution (simply freeze the probe mix after use)
Incubate in 1ml 50% hybridization buffer in PTW for Duration00:05:00 at TemperatureRoom temperature
Incubate in 300µl 100% hybridization buffer for Duration01:00:00 at Temperature37 °C on a heat block .

Careful when replacing media to not lose any samples. The buffer is very viscous; wait until samples settle down to the bottom of the tube.
1h 5m
Toxic
Meanwhile prepare the probe solution:
  • 1pmol of each probe mix (1µl of 1µM stock) in 250µl hybridization buffer
  • heat the probe solution to 37°C (for small volumes, we use the heat block the samples are already on; for larger volumes, we use a water bath)
Toxic
Replace the hybridization buffer with the prepared probe solution and incubate the samples DurationOvernight at Temperature37 °C .

Toxic
Overnight
Wash out the HCR probes:
Wash for Duration00:15:00 with 1ml pre-heated probe wash buffer at Temperature37 °C
Wash for Duration00:15:00 with 1ml pre-heated probe wash buffer at Temperature37 °C
Wash for Duration00:15:00 with 1ml pre-heated probe wash buffer at Temperature37 °C
Wash for Duration00:15:00 with 1ml pre-heated probe wash buffer at Temperature37 °C

Wash for Duration00:05:00 with 1ml 5X SSCT at TemperatureRoom temperature
Wash for Duration00:05:00 with 1ml 5X SSCT at TemperatureRoom temperature

From here on, the protocol progresses differently for each combination of methods
(HCR only, HCR & EdU, HCR & IHC, HCR, EdU & IHC)
Please select the step case below accordingly:
1h 10m
Toxic
Step case

HCR only
6 steps

This is the bare-bones protocol to just stain your samples with HCR.
Day 3: HCR probe detection
Day 3: HCR probe detection
1h 16m 30s
1h 16m 30s
HCR Amplification:

Wash with 1ml PTW for Duration00:05:00 at TemperatureRoom temperature
Equilibrate samples in 300µl amplification buffer for Duration00:30:00 at TemperatureRoom temperature

35m
Meanwhile:
Prepare the HCR hairpin mix.
  • Use 15 pmol per hairpin (5µl of 3µM stock) - move them to individual 1,5ml microcentrifuge tubes*
  • Heat the hairpins to Temperature95 °C for Duration00:01:30 by adding them to a pre-heated heatblock
  • Let the hairpins cool to TemperatureRoom temperature protected from light for Duration00:30:00
  • Mix the cooled hairpins with amplification buffer to a total volume of 250µl

* NOTE: for each amplifier, TWO different hairpins have to be used! (e.g., B1H1 and B1H2 are a pair of hairpins compatible with the B1 amplifier sequence) - heat those in separate tubes and pool only directly before adding them to the sample!

* NOTE: several probe pairs can be co-detected in one sample by using different sets of amplifiers with different fluorophores.
31m 30s
Incubate the samples in 250 µl hairpin mix DurationOvernight protected from light at TemperatureRoom temperature .

Note: amplification buffer is viscous; samples will eventually equilibrate and sink to the bottom of the tube, but careful pipetting is recommended to not lose any samples.

Day 4: Amplification Termination
Day 4: Amplification Termination
1h 25m
1h 25m
Terminate the amplification:
Wash in 1ml 5X SSCT for Duration00:05:00 at TemperatureRoom temperature
Wash in 1ml 5X SSCT for Duration00:05:00 at TemperatureRoom temperature
Wash in 1ml 5X SSCT for Duration01:00:00 at TemperatureRoom temperature
Wash in 1ml 5X SSCT for Duration00:05:00 at TemperatureRoom temperature
Wash in 1ml PTW for Duration00:05:00 at TemperatureRoom temperature shaking on rocking platform
Staining of nuclei (DAPI/ Hoechst) can be performed here by adding the dye directly to PTW and increasing the incubation time to Duration00:15:00 .
Wash in 1ml PTW for Duration00:05:00 at TemperatureRoom temperature shaking on rocking platform

1h 40m
Mount the samples:
Mounting strategies may differ based on sample type/ size.
For whole mount Platynereis blastemas or heads, the following works well:

Equilibrate the sample in a small volume of mounting medium (in our hands, SlowFade Diamond worked best)
Put four small pieces of "pattafix" or similar poster mounting clay (Blu-tack or similar) into the corners of the "mounting area" of a microscope slide to build a stage.
Put the sample in the middle of the stage, and add a small volume (~10µl) of mounting medium on top of it.

Gently press down a cover glass, using a second microscope slide to keep it even.