Aug 09, 2024

Public workspaceAn improved whole-mount immunofluorescence protocol for juvenile Amphimedon queenslandica V.1

DOI
dx.doi.org/10.17504/protocols.io.e6nvw1o2wlmk/v1
  • 1School of the Environment and Centre for Marine Science, The University of Queensland, Brisbane 4072, Australia
  • Sandie M Degnan: Corresponding author
Bin Yang
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DOI: dx.doi.org/10.17504/protocols.io.e6nvw1o2wlmk/v1
Protocol Citation: Bin Yang, Bernard M. Degnan, Sandie M Degnan 2024. An improved whole-mount immunofluorescence protocol for juvenile Amphimedon queenslandica. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1o2wlmk/v1
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: July 25, 2024
Last Modified: August 09, 2024
Protocol Integer ID: 104038
Keywords: whole-mount immunofluorescence, sponge, Amphimedon queenslandica, interferon regulatory factor 2
Funders Acknowledgements:
Australian Research Council
Grant ID: DP190102521
Australian Research Council
Grant ID: DP230102109
Gordon & Betty Moore Foundation
Grant ID: GMBF9352
Abstract
Sponges offer a unique perspective on animal evolution because of their simple body plan and phylogenetic position in the animal kingdom (Renard et al. 2018). However, established methods to investigate molecular and cellular processes in animal models are often underdeveloped in sponges. One such method is immunofluorescence, which has been successfully applied in multiple sponges but often restricted to specific commercial or tailored antibodies, sponge species and developmental contexts (e.g., Nakanishi et al. 2014; Schippers and Nichols 2018; Musser et al. 2021). Here we present an approach that improves on existing immunofluorescence protocols for Amphimedon queenslandica (Nakanishi et al. 2014; Sogabe et al. 2016) to detect subcellular localisation of the low abundance transcription factor interferon regulatory factor 2 (IRF2) in juvenile sponges; this detection could not be achieved using previously published methods nor using a range of other approaches that we trialled (Fig. 1). Specifically, we describe how sodium dodecyl sulfate (SDS) is introduced to reverse antigen masking that often appears after aldehyde-based fixation and to improve signal-to-noise ratios of immunostaining patterns (Shi et al. 2001; Salameh et al. 2018). A buffer containing PIPES, HEPES, EGTA and MgCl2 (PHEM) is also introduced in rehydration and immunostaining steps to enhance morphological preservation and subcellular detection of IRF2. These modifications yielded significantly improved results (Fig. 1), and are likely to assist in detecting low abundance proteins in other sponges.
Materials
Materials
  1. Costar tissue culture-treated 6 well plates. Note: it is necessary to use this specific brand to achieve successful larval settlement (Corning, catalog number: CLS3516)
  2. Prolong glass antifade mountant (Thermo Fisher Scientific, catalog number: P36980)
  3. Microscope slides (SAIL Brand, catalog number: 7103)
  4. Coverslips (Carl Zeiss, catalog number: 474030-9000-000)
  5. Transfer pipettes (Sigma-Aldrich, catalog number: Z135011)
  6. 0.22 μm filtered artificial seawater (Tropic Marin Pro-Reef, 10 kg packages)
  7. PFA+GA fixative: fresh (no more than 4 hours old) 4% paraformaldehyde and 0.05% glutaraldehyde in 0.22 μm filtered artificial seawater
  8. 30%, 50% and 70% ethanol
  9. PHEM buffer: 60 mM PIPES, 25 mM HEPES, 10 mM EGTA and 2 mM MgCl2; pH 6.9
  10. 1% sodium dodecyl sulfate (1% SDS)
  11. Bovine serum albumin (BSA; Sigma-Aldrich, catalog number: A8022)
  12. Blocking buffer: 1% BSA in PHEM buffer
  13. AqIRF2 polyclonal antibody raised against TRSGSSADEQEPERPER peptide in rabbit (GenScript Biotech)
  14. Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 647 (Invitrogen, catalog number: A21245)
  15. Hoechst 33342 (Thermo Fisher Scientific, catalog number: 62249)
Equipment and software
  1. Sunflower mini shaker (Grant-Bio, catalog number: PS-3D)
  2. Fluorescence stereomicroscope (Nikon SM225)
  3. Inverted fluorescence microscope (Nikon Eclipse Ti)
  4. Confocal microscope (Zeiss LSM 900)
  5. Fiji (Version 2.15.1)
Before start
Juvenile Amphimedon queenslandica are reared on coverslips to 4 days old in 6-well plates using filtered artificial seawater as previously described (Sogabe et al. 2016).
To achieve optimal preservation of the sponge samples, it is critical that PFA+GA fixative (see below) is prepared no more than 4 hours before being used for fixation.
Initial fixation
Initial fixation
Remove all filtered artificial seawater from a 6-well plate containing juveniles reared on coverslips (Sogabe et al. 2016) and add 0.5 mL of PFA+GA fixative into each well. Note: other fixatives trialled under the same conditions are less effective in preserving A. queenslandica juvenile morphology (Fig. 1).

Fig. 1. Fixation, rehydration and antigen retrieval methods and reagents trialled during the development of an optimised immunofluorescence protocol for Amphimedon queenslandica juveniles. The steps described in this report are in the solid colour boxes. Strikethrough text represents reagents that produced insufficient or poor results and thus are not included in the final protocol.

Gently swirl the 6-well plate for 5 seconds and immediately replace the initial fixative with 1 mL of fresh PFA+GA fixative.
Incubate juveniles in PFA+GA fixative on a slow nutator (10 rotations per minute) for 60 minutes at room temperature. Note: all subsequent wash steps in sections Dehydration and rehydration, Antigen retrieval and Immunostaining are conducted on the nutator at this rotation speed.
Dehydration and rehydration
Dehydration and rehydration
Remove all fixative from the wells and then wash and dehydrate fixed juveniles by stepping through 1 mL of 30%, 50% and 70% ethanol (each pre-chilled at -20 °C) for 5 minutes each. Note: dehydrated juveniles can be stored in 70% ethanol at -20 °C for up to 6 months.
Remove all ethanol from wells and then rehydrate dehydrated juveniles by washing them 4 times for 5 minutes each in 1 mL of PHEM buffer at room temperature. Note: other buffers either do not maintain morphology or are not compatible with the SDS antigen retrieval step (Fig. 1).
Antigen retrieval
Antigen retrieval
To reverse antigen masking that appears to occur after aldehyde-based fixation, incubate rehydrated juveniles in 1 mL of 1% SDS for 5 minutes.
Remove residual SDS from the juvenile samples by washing them 3 times for 5 minutes each in 1 mL PHEM buffer at room temperature. This maintains juvenile morphology, as evidenced by the arrangement of choanocytes and choanocyte chambers (Fig. 2).

Fig. 2. Choanocyte chambers in juveniles fixed with GA+PFA fixative, rehydrated with PHEM and then subjected to SDS antigen retrieval treatment. Spherical patterns of nuclei (blue) of choanocyte chambers (outlined by yellow dashed lines) in the internal cell layer of Hoechst-stained juveniles (outlined by white dashed lines). Photomicrograph from a Nikon fluorescence stereomicroscope. Scale bar, 30 μm.

Immunostaining
Immunostaining
Incubate juveniles in 1 mL of blocking buffer for 1 hour at room temperature on the bench without rotating.
Gently remove all the blocking buffer and then incubate juveniles in 0.5 mL of fresh blocking buffer containing 1 μL of the primary antibody (anti-AqIRF2 rabbit antiserum) at 4 °C overnight in the dark without rotating.
Wash immunolabelled juveniles in 1 ml of PHEM buffer 3 times for 5 minutes at room temperature on the nutator.
Incubate the juveniles in 0.5 mL of fresh blocking buffer containing 0.5 μL of the secondary antibody (Alexa Fluor 647 goat anti-rabbit IgG) for 30 minutes at room temperature in the dark without rotating.
Wash immunostained juveniles in 1 mL of PHEM buffer 3 times for 5 minutes at room temperature on the nutator.
Counterstain nuclei in the juveniles with 500 μL of 1 μg/mL Hoechst 33342 in PHEM buffer for 20 minutes at room temperature in the dark without rotating.
Repeat the washes 3 times for 5 minutes each in 1 mL of PHEM on the nutator, then mount the juveniles in Prolong Glass Antifade Mountant on microscope slides.
Capture images of the cellular localisation of nuclei and the target protein in these juveniles using a confocal microscope, and analyze these images using Fiji software (Schindelin et al. 2012) (Fig. 3).

Fig. 3. IRF2 immunostaining pattern in juvenile cells.Immunolocalisation of IRF2 (red) in cells in and near choanocyte chambers (outlined by dashed lines) in A. queenslandica juveniles. Apical microvilli of individual choanocytes are labelled along with nuclei in amoebocytes (yellow arrowheads) and archaeocytes (white arrowheads). Photomicrograph from a Zeiss LSM 900 confocal microscope. Scale bar, 10 µm.

Protocol references
Musser, J.M., Schippers, K.J., Nickel, M., Mizzon, G., Kohn, A.B., Pape, C., Ronchi, P., Papadopoulos, N., Tarashansky, A.J., Hammel, J.U., Wolf, F., Liang, C., Hernández-Plaza, A., Cantalapiedra, C.P., Achim, K., Schieber, N.L., Pan, L., Ruperti, F., Francis, W.R., Vargas, S., Kling, S., Renkert, M., Polikarpov, M., Bourenkov, G., Feuda, R., Gaspar, I., Burkhardt, P., Wang, B., Bork, P., Beck, M., Schneider, T.R., Kreshuk, A., Wörheide, G., Huerta-Cepas, J., Schwab, Y., Moroz, L.L., Arendt, D. (2021). Profiling cellular diversity in sponges informs animal cell type and nervous system evolution. Science 374: 717–723.

Nakanishi, N., Sogabe, S., Degnan, B.M. (2014). Evolutionary origin of gastrulation: insights from sponge development. BMC Biology 12: 26.

Renard, E., Leys, S. P., Wörheide, G., & Borchiellini, C. (2018). Understanding Animal Evolution: The added value of sponge transcriptomics and genomics: the disconnect between gene content and body plan evolution. BioEssays 40: 1700237.

Salameh, S., Nouel, D., Flores, C., Hoops, D. (2018). An optimized immunohistochemistry protocol for detecting the guidance cue Netrin-1 in neural tissue. MethodsX 5: 1–7.

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P., Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods 9: 676–682.

Schippers, K.J., Nichols, S.A. (2018). Evidence of signaling and adhesion roles for β-catenin in the sponge Ephydatia muelleri. Molecular Biology and Evolution 35: 1407–1421.

Shi, S.-R., Cote, R. J., Taylor, C. R. (2001). Antigen retrieval techniques: current perspectives. Journal of Histochemistry and Cytochemistry 49: 931–937.

Sogabe, S., Nakanishi, N., Degnan, B.M. (2016). The ontogeny of choanocyte chambers during metamorphosis in the demosponge Amphimedon queenslandica. EvoDevo 7: 1–13.