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      Fluorescent in situ hybridization in sponge (Ephydatia muelleri) tissues with tyramide signal amplification

      April Hill, Sally P Leys, Vanessa R Ho

      Published: 2024-02-09 DOI: 10.17504/protocols.io.j8nlkonkxv5r/v1
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      Abstract

      Cultured sponges such as Ephydatia muelleri (Demospongiae) have delicate tissues and are attached to coverslips, unlike many other tissue samples such as embryos that are typically free floating. This protocol also uses urea in place of formamide, which is not only safer, but is just as effective at reducing background signals and also improves signal detection.

      It is strongly recommended to have all the reagents prepared at least a day before they are needed. Large quantities of some solutions like PBS can be made at one time but some solutions such as the proteinase K solution are to be prepared fresh.

      Steps

      Tissue fixation and preparation

      1.

      Fix sponges in 4% paraformaldehyde in 1:4 Holtfreter's solution (HS; diluted 1 in 4) overnight

      2.

      Wash once in 1:4 HS

      Note
      All washes are 5 min unless otherwise indicated

      3.

      Dehydrate the tissues

      1. 25% ethanol (EtOH) and 75% 1:4 HS
      2. 50% EtOH and 50% 1:4 HS
      3. 75% EtOH and 25% 1:4 HS
      4. 100% EtOH
      3.1.

      Dehydrated tissues can be stored at -80ºC until ready to use.

      ISH Day 1a - tissue pre-treatment

      4.
      Setup for ISH day 1 in a dedicated RNA-clean space with RNA-clean supplies
      Setup for ISH day 1 in a dedicated RNA-clean space with RNA-clean supplies

      Rehydrate sponges - carefully and quickly transfer coverslips into the wells of the cell culture plates

      1. 100% EtOH
      2. 75% EtOH:25% phosphate buffered saline (PBS)
      3. 50% EtOH:50% PBS
      4. 25% EtOH: 75% PBS
      5. 100% PBS
      5.

      Quench endogenous peroxidases 15 min with 2% H2O2 in EtOH

      6.

      Rinse tissues

      1. PBS x2
      2. 2 min PBTw x3
      7.

      1 min proteinase K digestion

      8.

      Wash with Gly:PBTw solution x2 to stop digestion

      9.

      Acetylation

      1. 1% TEA:PBS x2 (Tube #1)
      2. 1% TEA:PBS + acetic anhydride (Tube #2) - 3 μL acetic anhydride per 4 mL 1%TEA:PBS
      3. 1% TEA:PBS + acetic anhydride (Tube #3) - 6 μL acetic anhydride per 4 mL 1% TEA:PBS
      10.

      Rinse with PBTw x2

      11.

      Post-fix in 3.7% paraformaldehyde (PFH) + 0.3% gluteraldehyde, at least 1 h at room temperature (RT)

      Note
      Can be left overnight in post-fix if neededUse this time to thaw hybridization buffer (HB) to RT and pre-heat a separate tube of HB if continuing past post-fix in the same day

      ISH Day 1b - pre-hybridization

      12.

      Wash with PBTw x5

      13.

      Wash with HB 10 min at RT

      14.

      Replace HB with fresh, pre-heated (55°C) HB, pre-hybridize at 55°C 2-3 h

      Note
      Use this time to prepare probes (closer to the end of pre-hyb period)

      ISH Day 1b - probe preparation and hybridization

      15.

      Dilute probe (we use 1:1000; 3 μL 50 ng/μL probe in a final volume of 3 mL HB)

      1. in labeled 1.5 mL Eppendorf tubes, add 3 μL probe to 0.97 mL pre-warmed (55°C) HB
      2. Make sure the lid is shut tight
        Note
        Probe template synthesized using gBlocks Gene Fragments (Integrated DNA Technologies)Reverse primer design includes T7 tail for RNA polymerase bindingPCR amplification of gBlocks using Phusion HF polymerasePCR thermocycler melting temp is 54ºC$$PCR clean up using MinElute column (Qiagen) according to manufacturer's instructionsRiboprobe synthesisMEGAscript T7 transcription kit (Thermo Fisher)DIG-labeling mix (Roche)LiCl2 precipitation and EtOH extraction
      15.1.

      Immediately denature probe after adding to Eppendorf tube by boiling at 80°C for 10 min

      • Replace the pre-hyb HB with 2 mL fresh, pre-warmed HB in the wells in the meantime
      15.2.

      Quickly add probe (final well volume 3 mL)

      Note
      Dry off the tube with a kimwipe before openingRemember to label the wells to the corresponding probe

      16.

      Hybridize 16-72 h at 55°C

      Sponges on coverslips in a 6-well dish prior to hybridization
      Sponges on coverslips in a 6-well dish prior to hybridization

      Note
      24-48 h is optimal, 36 h is usually most convenientuse a clean airtight container such as a plastic snap-lock container (wiped with RNase Zap or equivalent) and a damp kimwipe (wetted with leftover HB works great) to maintain humidity and prevent drying out/urea crystallizationincubate the day 2 wash solutions at the same time so they are ready to go

      ISH Day 2 - post-hybridization stringency washes

      17.
      Sponges after hybridization often detach from the coverslips. These were transferred to fresh HB in a 24-well dish using a sterile, trimmed pipette tip to prevent damaging the tissues. Make sure to note which probes and sponges are which in the new culture plate.
      Sponges after hybridization often detach from the coverslips. These were transferred to fresh HB in a 24-well dish using a sterile, trimmed pipette tip to prevent damaging the tissues. Make sure to note which probes and sponges are which in the new culture plate.

      Stringency washes - at 55°C

      1. 30 min fresh HB
      2. 20 min PHB1
      3. 20 min PHB2
      4. 20 min PHB3
      5. 20 min 2X SSC
        Note
        Preheat all solutionPHB1 - 75% PHB: 25% 2X SSC pH 7.0PHB2 - 50% PHB: 50% 2X SSC pH 7.0PHB3 - 25% PHB: 75% 2X SSC pH 7.0
      17.1.

      Stringency washes at RT

      1. 50% 2X SSC: 50% MAB 10 min
      2. 100% MAB x3 (5 min)
      18.

      Block 1 hr blocking buffer (bb) on rocker at RT

      19.

      Incubate overnight in antibody (anti-DIG-POD) at 4°C on rocker

      1. Make the stock at 1:500 dilution in bb
      2. Replace half the bb in the well from the previous step with the anti-DIG-POD for final antibody dilution of 1:1000

      ISH Day 3

      20.

      Wash 20 min MAB x5

      21.

      Wash 5 min TNT buffer x3

      ISH Day 3 - signal development - photosensitive steps; keep dark

      22.

      Incubate in 250 μL 1:50 TSA Plus working solution 20 min at RT

      Note
      This was calculated for use in 24-well plates, follow manufacturer's instructions for determining amounts needed

      23.

      Stop reaction

      1. Wash 5 min TNT buffer x3 on rocker
      2. Rinse with PBS
      24.

      Incubate in Hoechst 33342 (1:1000) at 15 min RT for nuclear counterstaining

      Safety information
      Handle with gloves. As a cell-permeant DNA dye, Hoechst 33342 can be potentially carcinogenic/mutagenic

      25.

      Rinse 5 min PBS X4

      Note
      May be stored at 4°C in the dark for a few days (eg. over the weekend) before mounting

      26.

      Mount and store tissue samples

      1. Clear tissues in Mowiol + DABCO anti-fade agent
      2. Mount on microscope slide
      3. Seal with nail polish and let dry
      4. Store at 4°C in the dark
        Single flattened sponge mounted on a microscope slide. To secure the glass coverslip scrape a small fragment of plasticine with each corner of the coverslip and gently press onto the sponge, avoiding air bubbles and shifting the sponge around.
        Single flattened sponge mounted on a microscope slide. To secure the glass coverslip scrape a small fragment of plasticine with each corner of the coverslip and gently press onto the sponge, avoiding air bubbles and shifting the sponge around.

      Note
      cut the tip off a P1000 pipette tip to transfer sponges without damaging the tissuesfine tip forceps and a 0.5 cm stab pen blade are helpful for flattening the sponge as it often curls up on itself

      M2 Silicatein mRNA expression (green) in a 4-days post-hatch (stage 3 juvenile) Ephydatia muelleri specimen viewed with a standard FITC filter and merged with the image of Hoechst 33342-stained nuclei (blue) viewed with a DAPI filter. Scale bar = 0.5 mm
      M2 Silicatein mRNA expression (green) in a 4-days post-hatch (stage 3 juvenile) Ephydatia muelleri specimen viewed with a standard FITC filter and merged with the image of Hoechst 33342-stained nuclei (blue) viewed with a DAPI filter. Scale bar = 0.5 mm

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