TITLE: SHInE - Simultaneous HCR, Immunohistochemistry, Nuclear staining and EdU
AUTHORS: Aida Coric, Alexander W. Stockinger, Petra Schaffer, Dunja Rokvic, Kristin Tessmar-Raible, Florian Raible

[DESCRIPTION]
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).



[STEPS]
SECTION: Optional: EdU incubation
2. Optional: Chase out EdU with 3 millimolar (mM)  thymidine for 60 min ; then replace thymidine solution with fresh ASW and incubate for desired chase length

SECTION: Optional: EdU incubation
1. Incubate worms with 10 micromolar (µM) EdU in artificial sea water (ASW) (EdU aliquots in both DMSO and H2O work fine) for your desired pulse length.

SECTION: Day 1: Fixation, Dehydration
3. 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 on ice 

Note: the protocol is also compatible with whole animals, but will require larger volumes and more reagent accordingly.
SECTION: Day 1: Fixation, Dehydration
4. 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 60 minat Room 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. 
SECTION: Day 1: Fixation, Dehydration
5. 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 3 min 
50% MeOH in PTW 3 min 
75% MeOH in PTW3 min 
Wash in100% MeOH 1 min 
Wash in100% MeOH 1 min 
SECTION: Day 1: Fixation, Dehydration
6. Store samples at -20° C for at least   . Longer storage is possible (several months in our hands, but even longer storage might be possible).

SECTION: Day 2: Rehydration, digestion, probe hybridization
7. Re-hydrate samples through a series of decreasing MeOH concentrations in 1X PTW on ice. 
75% MeOH in PTW 3 min 
50% MeOH in PTW3 min 
25% MeOH in PTW3 min 
Wash 2x in 1X PTW3 min 



SECTION: Day 2: Rehydration, digestion, probe hybridization
8. Perform proteinase K treatment with 1ml of Proteinase K in PTW according to table below, at Room temperature .
SECTION: Day 2: Rehydration, digestion, probe hybridization
8.1. 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.
 
 

 
SECTION: Day 2: Rehydration, digestion, probe hybridization
8.2. Briefly rinse 2x with 1 ml of glycine wash buffer to stop digest. Work on ice from here on.
Wash once for 5 min with 1x PTW. 


SECTION: Day 2: Rehydration, digestion, probe hybridization
8.3. Post-fix in 1mL 4%PFA diluted in 1xPTW for 20 min shaking while still on ice.
SECTION: Day 2: Rehydration, digestion, probe hybridization
8.4. Wash samples 2x 5 min in 1ml PTW.

SECTION: Day 2: Rehydration, digestion, probe hybridization
9. Optional: tissue clearing
If clearing with DEEP-CLEAR is wanted, perform it at this step. Worm tissue can be cleared at 37 °C for 15 min at 300 rpm on a heat block. Clearing time should be optimised for different tissues. Wash the samples in PTW thoroughly after clearing (3x 5 min ).

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)
SECTION: Day 2: Rehydration, digestion, probe hybridization
10. 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)
SECTION: Day 2: Rehydration, digestion, probe hybridization
10.1. Incubate in 1ml 50% hybridization buffer in PTW for 5 min at Room temperature 
Incubate in 300µl 100% hybridization buffer for 60 min  at 37 °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. 
SECTION: Day 2: Rehydration, digestion, probe hybridization
10.2. 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)
SECTION: Day 2: Rehydration, digestion, probe hybridization
10.3. Replace the hybridization buffer with the prepared probe solution and incubate the samples   at 37 °C.

SECTION: Day 3: Washes
11. Wash out the HCR probes:
Wash for 15 min with 1ml pre-heated probe wash buffer at 37 °C 
Wash for 15 min with 1ml pre-heated probe wash buffer at 37 °C 
Wash for 15 min with 1ml pre-heated probe wash buffer at 37 °C 
Wash for 15 min with 1ml pre-heated probe wash buffer at 37 °C 

Wash for 5 min with 1ml 5X SSCT at Room temperature
Wash for 5 min with 1ml 5X SSCT at Room 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:
SECTION: Day 3: HCR probe detection
12. HCR Amplification:

SECTION: Day 3: HCR probe detection
12.1. Wash with 1ml PTW for 5 min at Room temperature 
Equilibrate samples in 300µl amplification buffer for 30 min at Room temperature 

SECTION: Day 3: HCR probe detection
12.2. 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 95 °C for 90 s by adding them to a pre-heated heatblock
Let the hairpins cool to Room temperature protected from light for 30 min 
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. 
SECTION: Day 3: HCR probe detection
12.3. Incubate the samples in 250 µl hairpin mix   protected from light at Room 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. 

SECTION: Day 4: Amplification Termination
13. Terminate the amplification:
Wash in 1ml 5X SSCT for 5 min at Room temperature 
Wash in 1ml 5X SSCT for 5 min at Room temperature 
Wash in 1ml 5X SSCT for 60 min at Room temperature 
Wash in 1ml 5X SSCT for 5 min at Room temperature 
Wash in 1ml PTW for 5 min at Room 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 15 min .
Wash in 1ml PTW for 5 min at Room temperature shaking on rocking platform

SECTION: Day 4: Amplification Termination
14. 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.
SECTION: Day 3: EdU click chemistry; HCR probe detection
12. EdU detection with click chemistry:
SECTION: Day 3: EdU click chemistry; HCR probe detection
12.1. Wash samples in PTW for 5 min at Room temperature (shaking on rocking platform)
Wash samples in PTW for 5 min at Room temperature (shaking on rocking platform)
SECTION: Day 3: EdU click chemistry; HCR probe detection
12.2. Meanwhile, prepare the "click cocktail" according to manufacturer's protocol (see below):

Note on reaction volume: depending on sample size, this could be enough for 1-2 1,5ml tubes; the staining reaction is extremely robust and strong in our hands, so a small volume (covering the samples fully) should be sufficient.

For 500µl click cocktail, mix (in order):
430µl 1X reaction buffer
20µl CuSo4*
1,2µl Alexa Fluor Azide
50µl reaction buffer additive (Note: this is stored at 10X concentration! Dilute directly prior to use)


* for samples with endogenous fluorescence, copper ions might be problematic. There is an alternative click reaction kit called "click-it plus" which works well with these samples and is also compatible with SHINE.
SECTION: Day 3: EdU click chemistry; HCR probe detection
12.3. Incubate with click-cocktail for 30 min at Room temperature protected from light.
SECTION: Day 3: EdU click chemistry; HCR probe detection
13. HCR Amplification:
SECTION: Day 3: EdU click chemistry; HCR probe detection
13.1. Wash with 1ml PTW for 5 min at Room temperature 
Wash with 1ml PTW for 5 min at Room temperature 
Wash with 1ml PTW for 5 min at Room temperature 
Equilibrate samples in 300µl amplification buffer for 30 min at Room temperature 
SECTION: Day 3: EdU click chemistry; HCR probe detection
13.2. 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 95 °C for 90 s by adding them to a pre-heated heatblock
Let the hairpins cool to Room temperature protected from light for 30 min 
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!
SECTION: Day 3: EdU click chemistry; HCR probe detection
13.3. Incubate the samples in hairpin mix   protected from light at Room temperature 
SECTION: Day 4: Amplification Termination; mounting
14. Wash out the amplification mix:
Wash in 1ml 5X SSCT for 5 min at Room temperature 
Wash in 1ml 5X SSCT for 5 min at Room temperature 
Wash in 1ml 5X SSCT for 60 min at Room temperature 
Wash in 1ml 5X SSCT for 5 min at Room temperature 

Wash in 1ml PTW for 5 min at Room 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 15 min . Hoechst 33342 at a concentration of 10µg/ml works well in our samples. 
Wash in 1ml PTW for 5 min at Room temperature shaking on rocking platform

SECTION: Day 4: Amplification Termination; mounting
15. 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 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 (see picture below).
SECTION: Day 3: HCR probe detection; primary antibody
12. HCR amplification & primary antibody:
SECTION: Day 3: HCR probe detection; primary antibody
12.1. Wash with 1ml PTW for 5 min at Room temperature 
Wash with 1ml PTW for 5 min at Room temperature 
Equilibrate samples in 300µl amplification buffer for 30 min at Room temperature 
SECTION: Day 3: HCR probe detection; primary antibody
12.2. 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 95 °C for 90 s by adding them to a pre-heated heatblock
Let the hairpins cool to Room temperature protected from light for 30 min 
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!
SECTION: Day 3: HCR probe detection; primary antibody
12.3. Add the primary antibody to the cooled and pooled hairpin mix (after addition of the amplification buffer). 

Note:
In our hands, a primary antibody dilution of 1:200 for commercial monoclonal antibodies worked well.
The dextrane sulfate in the amplification buffer can cause binding issues with antibodies. Adjusting its concentration could help, but was not necessary for the antibodies we used. 
SECTION: Day 3: HCR probe detection; primary antibody
12.4. Incubate the samples in hairpin + antibody mix   protected from light at Room temperature 
SECTION: Day 4: Amplification Termination; secondary antibody
13. Wash in 1ml 5X SSCT for 5 min at Room temperature 
Wash in 1ml 5X SSCT for 5 min at Room temperature 
Wash in 1ml 5X SSCT for 60 min at Room temperature 
Wash in 1ml 5X SSCT for 5 min at Room temperature 

Wash in 1ml PTW for 5 min at Room temperature shaking on rocking platform
Wash in 1ml PTW for 5 min at Room temperature shaking on rocking platform
SECTION: Day 4: Amplification Termination; secondary antibody
14. Incubate with secondary antibody:
Incubate in secondary antibody (500µl, 1:500 dilution) in PTW   at 4 °C 

Note: The ideal antibody concentration might be sample- and antibody dependent.

SECTION: Day 4: Amplification Termination; secondary antibody
14.1. Staining of nuclei (DAPI/Hoechst) can be performed  during this step.
Hoechst 33342 at a concentration of 10µg/ml works well in our samples. 
SECTION: Day 5: Washes; Mounting
15. Wash out secondary antibody:
Wash in 1ml PTW for 15 min at Room temperature shaking on rocking platform
Wash in 1ml PTW for 15 min at Room temperature shaking on rocking platform
SECTION: Day 5: Washes; Mounting
16. 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 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 (see picture below).
SECTION: Day 3: EdU click reaction; HCR probe detection; primary antibody
12. EdU decection with Click Chemistry:
SECTION: Day 3: EdU click reaction; HCR probe detection; primary antibody
12.1. Wash samples in PTW for 5 min at Room temperature (shaking on rocking platform)
Wash samples in PTW for 5 min at Room temperature (shaking on rocking platform)
SECTION: Day 3: EdU click reaction; HCR probe detection; primary antibody
12.2. Meanwhile, prepare the "click cocktail" according to manufacturer's protocol (see below):

Note on reaction volume: depending on sample size, this could be enough for 1-2 1,5ml tubes; the staining reaction is extremely robust and strong in our hands, so a small volume (covering the samples fully) should be sufficient.

For 500µl click cocktail, mix (in order):
430µl 1X reaction buffer
20µl CuSo4*
1,2µl Alexa Fluor Azide
50µl reaction buffer additive (Note: this is stored at 10X concentration! Dilute directly prior to use)


* for samples with endogenous fluorescence, copper ions might be problematic. There is an alternative click reaction kit called "click-it plus" which works well with these samples and is also compatible with SHINE.
SECTION: Day 3: EdU click reaction; HCR probe detection; primary antibody
12.3. Incubate with click cocktail for 30 min at Room temperature protected from light.
SECTION: Day 3: EdU click reaction; HCR probe detection; primary antibody
13. HCR amplification & primary antibody:
SECTION: Day 3: EdU click reaction; HCR probe detection; primary antibody
13.1. Wash with 1ml PTW for 5 min at Room temperature 
Wash with 1ml PTW for 5 min at Room temperature 
Wash with 1ml PTW for 5 min at Room temperature 
Equilibrate samples in 300µl amplification buffer for 30 min at Room temperature 
SECTION: Day 3: EdU click reaction; HCR probe detection; primary antibody
13.2. 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 95 °C for 90 s by adding them to a pre-heated heatblock
Let the hairpins cool to Room temperature protected from light for 30 min 
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!
SECTION: Day 3: EdU click reaction; HCR probe detection; primary antibody
13.3. Add the primary antibody to the cooled and pooled hairpin mix (after addition of the amplification buffer). 

Note:
In our hands, a primary antibody dilution of 1:200 for commercial monoclonal antibodies worked well.
The dextrane sulfate in the amplification buffer can cause binding issues with antibodies. Adjusting its concentration could help, but was not necessary for the antibodies we used. 
SECTION: Day 3: EdU click reaction; HCR probe detection; primary antibody
13.4. Incubate the samples in hairpin + antibody mix   protected from light at Room temperature 
SECTION: Day 4: Amplification termination; secondary Antibody; Mounting
14. Terminate the amplification:
Wash in 1ml 5X SSCT for 5 min at Room temperature 
Wash in 1ml 5X SSCT for 5 min at Room temperature 
Wash in 1ml 5X SSCT for 60 min at Room temperature 
Wash in 1ml 5X SSCT for 5 min at Room temperature 
Wash in 1ml PTW for 5 min at Room temperature shaking on rocking platform
Wash in 1ml PTW for 5 min at Room temperature shaking on rocking platform
SECTION: Day 4: Amplification termination; secondary Antibody; Mounting
15. Incubate with secondary antibody:
Incubate in secondary antibody (500µl, 1:500 dilution) in PTW   at 4 °C 

Note: The ideal antibody concentration might be sample- and antibody dependent.
SECTION: Day 4: Amplification termination; secondary Antibody; Mounting
15.1. Staining of nuclei (DAPI/Hoechst) can be performed during this step.
Hoechst 33342 at a concentration of 10 µg/ml works well in our samples. 

SECTION: Day 5: Washes; mounting
16. Wash out secondary antibody:
Wash in 1ml PTW for 15 min at Room temperature shaking on rocking platform
Wash in 1ml PTW for 15 min at Room temperature shaking on rocking platform
SECTION: Day 5: Washes; mounting
17. 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 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 (see picture below).