Paraformaldehyde (pfa)

After 96 h of culture, some samples were immersed in Rembaum solution [66 (link)] for 1 h then rinsed three times with demineralized water. They were observed using SEM (Philips XL30 ESEM-FEG, Eindhoven, The Netherlands; quanta FEG 250, FEI, Hillsboro, OR, USA; Hitachi S-3400N, Tokyo, Japan) after gold coating.
Others were stained to observe the alignment of the actin filaments using fluorescence microscopy. Briefly, samples were fixed in paraformaldehyde (Agar Scientific, Stansted, UK), immersed for 45 min in a rhodamine/phalloidin solution (5 units/mL, Invitrogen, Waltham, MA, USA) then rinsed with PBS. The nuclei of the cells were also stained with DAPI (1 g/L, Invitrogen, Waltham, MA, USA). Samples were then observed (Leica Microsystems, Wetzlar, Germany) with excitation and emission wavelengths of 540/565 nm (rhodamine/phalloidin) and 358/461 nm (DAPI).

A final concentration of 2.5% glutaraldehyde (TAAB) was added to the culture suspension. Cells were pelleted and resuspended in a primary fixative containing 2.5% glutaraldehyde, 2% paraformaldehyde (Agar Scientific), and 0.1% tannic acid (TAAB) in 0.1 M phosphate buffer, pH 7.0 (Sigma-Aldrich). Cells were fixed for 2 h at room temperature. Pellets were washed with 0.1 M phosphate buffer (pH 7.0) and postfixed in 1% osmium tetroxide (Agar Scientific) in 0.1 M phosphate buffer (pH 7.0) for 1 h at room temperature. Samples were rinsed and stained en bloc for 40 min in 2% aqueous uranyl acetate (TAAB), dehydrated in an ascending acetone series (Fisher Scientific), and embedded in hard formulation Agar 100 resin (Agar Scientific). 70-nm thin sections were made and stained using lead citrate for 5 min, followed by three washes with MilliQ water. Images were captured using a Hitachi H-7650 transmission electron microscope.

Tissue was processed for electron microscopy as previously described (Czopka and Lyons, 2011 (link)). Briefly, 4 dpf larvae were anaesthetised with tricaine and chemically fixed with microwave stimulation using a solution of 2% glutaraldehyde, 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (Agar Scientific, Essex, UK). Embryos subsequently underwent secondary fixation with microwave stimulation in 2% osmium tetroxide in 0.1 M sodium cacodylate/imidazole (Agar Scientific) followed by en bloc stain in saturated uranyl acetate (~8%, w/v, in water) (TAAB Laboratories Equipment Ltd., Berkshire, UK). Samples were then progressively dehydrated in a series of ethanol and acetone washes before being embedded in Embed-812 resin (Electron Microscopy Sciences, Hatfield, PA). Ultra-thin sections (70 nm thickness) were cut around somite 15 using a Reichert Jung Ultracut microtome (Leica Microsystems, Wetzlar, Germany) and stained using uranyl acetate (TAAB Laboratories Equipment Ltd.) and Sato lead stain (see (Czopka and Lyons, 2011 (link)) for detailed protocol). Stained sections were imaged using a Jeol JEM-1400 Plus transmission electron microscope (JEOL USA, Inc., Peabody, MA) at 8600x magnification. Individual tiles were automatically aligned using the PhotoMerge tool in Adobe Photoshop CC (Adobe Systems Inc., San Jose, CA) and quantified manually using Fiji (ImageJ).

Mouse eyes were fixed with 2% paraformaldehyde (Agar Scientific Ltd. Cambridge, UK) for 2 hr. After paraffin embedding the eyeballs were cut into 5 μm thick sections and mounted on charged glass slides. Slides were de‐paraffinized and subjected to citrate‐based antigen retrieval. Paraffin sections were retreated with DAKO high pH antigen retrieval system (DAKO, Carpinteria, CA) using a domestic 600 kW microwave oven. Nonspecific antibody binding was blocked by incubating sections in 4% BSA, followed by 10% nonimmune goat serum (Zymed Corp., San Francisco, CA). Primary antibody was applied at a 1:200 to 400 dilutions overnight at room temperature. Sections then were incubated with secondary antibody for 30 min. The localization of target proteins was demonstrated with pre‐diluted streptavidin‐horseradish peroxidase (Zymed, UK) and 0.05% 3, 3‐diaminobenzidine in TBS, with H₂O₂ as the substrate. All sections were counterstained lightly with hematoxylin.

Microdissected cartilage from mice was fixed using 2.5% glutaraldehyde + 4% paraformaldehyde (stocks solutions both from Agar Scientific) in 0.1 M 1,4 Piperazine bis (2-ethanosulfonic acid) (Sigma-Aldrich) buffer at pH 7.2 at room temperature for 1–2 h, then at 4 °C until further processing. Samples were prepared as described previously [10] and imaged on an FEI company Tecnai 12 TEM with a Gatan US1000 camera. Thereafter, autophagosomes from images were counted from cells.

Coverslips were removed from the Maximow slide assemblies, placed in 35-mm diameter plastic Petri dishes, washed once with HBHBSS, and incubated with control medium or medium containing the inhibitor to be tested. Inhibitors were obtained or prepared as stock concentrates (usually at a concentration 100–1,000× greater than that required) in dimethyl sulfoxide (DMSO) or H₂O. An equivalent volume of DMSO or H₂O was added to the control medium. After 5–48 hours incubation at 37°C, cultures were fixed in 4% paraformaldehyde (Agar Scientific, Essex, UK) in 0.1 M sodium phosphate buffer, pH 7.4, for 1 hour at room temperature, and then rinsed three times in phosphate-buffered saline (PBS).

Testes were dissected in 1X PBS, fixed for 30 min to 1 h in 4% paraformaldehyde (Agar Scientific, UK)/1X PBS, washed in PBS/0.1% TX-100 for at least 1 h and incubated overnight in Vectashield with DAPI (Vector Labs) before mounting.
For phalloidin staining to visualise actin cones, testes were dissected, fixed and washed as above then incubated with TRITC-phalloidin for 45 min at room temperature. Samples were extensively washed in 1X PBS/0.3% TX-100 before addition of the mountant.