Sp5ii aobs

FISH staining for visualization of S. marcescens was performed as previously described (54 (link)) using a probe S. marcescens Alexa Fluor488 designed by MetaSystems Indigo GmbH, Altusscheim, Germany, which uses a next generation FISH beacon-based technology. A bacterial universal Texas red probe was applied for dual staining. Confocal microscopy was performed on a Leica SP5 II AOBS tandem scanner spectral confocal system on a DMI6000 microscope and controlled by LASAF software (v2.8.3).

For mPS-PI, acquisitions were done with a Zeiss LSM 710 confocal microscope as described previously [53 (link)]. For cell wall and nucleus staining, excitation wavelength for DAPI was 405 nm and emission was collected from 410 to 460 nm. For Direct Red 23, the excitation wavelength was 561 nm and emission was collected from 565 to 720 nm. In all cases, fluorescence signals were recorded using a 40x objective and digitized as 8-bit 3D image stacks with a near-to-optimal voxel size of 0.17×0.17×0.35 μm³.
Image acquisition of leaf epidermal cells was performed on a Leica SP 5II AOBS tandem HyD confocal laser-scanning microscope equipped with a Plan APOX NA1.25 oil immersion objective. GFP was excited at 488 nm by a diode laser and detected between 500 and 540 nm. RFP was excited at 561 nm and detected between 580 and 640 nm. Time-lapse images were acquired every 30 mn for several hours to detect cell divisions.

For confocal imaging, larvae were mounted ventrally in 1% LMP agarose (16520050, Thermofisher, MA, USA) and imaged using a Leica SP5‐II AOBS tandem scanner confocal microscope attached to a Leica DMI 6000 inverted epifluorescence microscope and oil immersion 20× or 40× objectives. The microscope was located in the Wolfson Bioimaging Facility, Bristol and run using Leica LAS AF software (Leica, Germany). Maximum projection images were assembled using LAS AF Lite software (Leica) and Fiji.⁶⁰

Live larvae were imaged at 4 dpf and 5 dpf and were anesthetised in 0.1 mg mL⁻¹ MS222 under a fluorescent stereomicroscope (Leica). For Lightsheet fluorescence microscopy (Zeiss Z.1), 3 dpf larvae were embedded in 1% low-melting point agarose and were imaged for up to 6 h with an acquisition frequency of 15 min. Immunohistochemistry and ISH samples (n ≥ 4) were imaged using a confocal laser scanning microscopy (Leica, Buffalo Grove, IL, USA, SP5II AOBS attached to a Leica DM I6000 inverted epifluorescence microscope) using a 10× PL APO CS, 20× HC PL APO CORR or 40× PL APO CS (4.0, 0.7 and 1.3 numerical aperture respectively) lenses. The larvae were mounted laterally or ventrally in 1% low melting point agarose (dissolved in Danieau’s solution) before imaging. Images were processed using Fiji [38 (link)] and linear brightness and contrast adjustments were applied. All IHC and ISH images are representative full projection images of confocal stacks. Three-dimensional (3D) volume renders were generated in Imaris software package (Oxford Instruments) of the operculum and Smad9⁺ population of cells at the dorsal tip of the operculum. Thresholds were maintained between 40–60% and the volume function was used to measure the population of Smad9⁺ cells.

BMPre:GFP (Tg(5xBMPRE‐Xla.Id3:GFP))27 and sp7:GFP (Tg(Ola.sp7:NLS‐GFP))28 transgenic fish (in London AB background) were housed and maintained in standard conditions.29, 30 Experiments were approved by the University of Bristol Animal Welfare and Ethical Review Body (AWERB) and performed in accordance with a UK Home Office project license. Developmentally staged larvae (after euthanization in MS222) were fixed in 4% paraformaldehyde (1 hour), dehydrated to 100% methanol, and stored at −20°C before staining. Immunolabeling was as previously described.31 Primary antibodies were anti‐Smad9 (rabbit polyclonal, Abcam, Cambridge, MA, USA, ab96698) used at a 1/100 dilution and anti‐GFP (chicken polyclonal, Abcam, ab13970) used at a 1/200 dilution in blocking buffer (5% horse serum). Secondary antibodies were used (A21206 and A11041, Invitrogen, Carlsbad, CA, USA) in a 1/400 dilution and samples incubated with DAPI (Sigma‐Aldrich, St. Louis, MO, USA, 1/1000 dilution) to visualize nuclei. Samples were mounted in 1% low melting point agarose and imaged with a confocal laser scanning microscope (Leica, Buffalo Grove, IL, USA, SP5II AOBS attached to a Leica DM I6000 inverted epifluorescence microscope) using a 40× PL APO CS (1.3 numerical aperture) lens. Images were processed and color balanced in Fiji.32