Permount mounting medium

Histological procedures followed ref. ⁶³ . Specifically, primary branches were collected from the central third of panicles 12 and 16 d after heading and fixed in FAA (37% formaldehyde: ethanol: H₂O: acetic acid = 10:50:35:5), followed by a dehydration series in 50%, 70%, 85%, 95%, 100%, 100% and 100% ethanol and 25%, 50%, 75%, 100%, 100% and 100% Histo-Clear (National Diagnostics) series with ethanol as solvent. Paraplast (Leica Biosystems) was then added to each vial of samples and kept overnight, heated at 42 °C and placed in a 60 °C oven. The solution was replaced with molten Paraplast twice a day for 3 d. Samples were then embedded in paraffin using a Leica EG1150 tissue embedder, sectioned in 10-µm serial slices with a Leica RM2255 automated microtome and mounted on microscope slides at 42 °C on a Premiere XH-2001 Slide Warmer. Sections were then deparaffinized, rehydrated, stained with 0.05% (wt/vol) toluidine blue O for 1.5 min and then rinsed with water, dehydrated in ethanol, cleared with xylene and mounted with Permount Mounting Medium (Electron Microscopy Sciences). Images were taken using a Leica DM750 LED Biological Microscope with ICC50 camera module and Leica Acquire version 2.0 software. Experiments were repeated on three independent plants of each genotype.

Unfixed brains of 2 month old male mice were processed using the FD Rapid Golgi stain kit (FD Neurotechnologies, Columbia, MD). Halved brains underwent a 14 day impregnation period in kit solution A&B, followed by three 24-hour rinses in solution C with gentle shaking. Brains were flash frozen using isopentane and dry ice. Two-hundred micron sagittal sections were taken on a sliding microtome. Sections were mounted onto gelatin-coated slides and dried for 7 days. Slides were developed according to manufacturer’s instructions and coverslipped with Permount mounting medium (Electron Microscopy Sciences, Hatfield, PA). Neurons were imaged on a Zeiss Imager M.2 microscope and traced using Neurolucida software (MBF Bioscience, Williston, VT). For layer V neurons, dendritic spines were counted along 50–100 micron sections of the apical dendrite, starting at the base of the soma. For layer II/III neurons, dendritic spines were counted along the secondary basal dendrites. Dendritic spine density was calculated for three-to-five neurons per animal, three-to-four animals per group.

Room temperature cryosections were fixed in Bouin’s Fixative (Polysciences) for 20 minutes. Slides were rinsed in cold tap water for five minutes and then stained in Weigert’s Iron Hematoxylin working solution (Polysciences) for five seconds. Slides were rinsed in cold tap water for two minutes and then stained in Beibrich Scarlett-Acid Fuchsin Solution (Polysciences) for 15 seconds. Slides were rinsed in deionized water (DI) until the water running off the slide was clear. Phosphotungstic/Phosphomolybdic acid (Polysciences) was added directly to the tissue sections, incubated for ten minutes, and then drained onto a Kimwipe. Slides were stained in Aniline Blue (Polysciences) for 30 seconds and rinsed in DI three times for 30 seconds. 1% Acetic Acid (Polysciences) was pipetted directly onto the sections for one minute, and then rinsed in DI. Slides were dehydrated in 95% and 100% ethanol for two minutes each, cleared in xylene for two minutes, and then mounted with Permount mounting medium (Electron Microscopy Sciences).

Adult flies were anesthetized with CO₂ and heads were cut into half to ensure proper penetration of the fixative. The samples were put into freshly made fixative containing 2.5% glutaraldehyde, 2% paraformaldehyde and 0.05% Triton X-100 in 0.1M sodium cacodylate buffer (pH 7.2) on rotator for at least 4 hours until all fly eyes were sunk to the bottom of the tube, then change to same fixative without Triton and continue fixed at 4°C for 4 days on rotator. After washing, the fly eyes were post fixed in 1% OsO₄ for 1.5 hour, dehydrated in a series of ethanol solutions (30%, 50%, 70%, 85%, 95%, 100%), followed by two rinses with propylene oxide and embedded in EMbed812 epoxy resin (Electron Microscopy Sciences, Hatfield, PA). 500nm thick semi-thin sections were cut, mounted on glass slide and baked on hot plate overnight at 60°C. The sections are stained with 0.1% Toluidine blue, dried on hot plate and cover-slipped with Permount mounting medium (Electron Microscopy Sciences, Hatfield, PA) for light microscopy. 70nm ultra-thin sections were cut and mounted on formvar coated slot grids and stained with uranyl acetate and lead citrate. Imaging was performed by an electron microscope (CM12, FEI, Eindhoven, The Netherlands) at 120 kV, and recorded digitally using a camera system (Gatan 4k x 2.7K) with software Digital Micrograph (Gatan Inc., Pleasanton, CA).

Room temperature cryosections were rehydrated in 1X PBS for five minutes. Slides were rinsed under running tap water for one minute prior to staining. Slides were then stained with Hematoxylin 7211 (Richard-Allan Scientific) for ten minutes and rinsed under tap water for one minute and 30 seconds. Slides were then stained with Eosin-Y alcoholic (Richard-Allan Scientific) for three minutes and rinsed under tap water for one minute. The slides were then incubated in 100% ethanol for three minutes, allowed to air dry briefly, and mounted with Permount mounting medium (Electron Microscopy Sciences).

Room temperature cryosections were rehydrated in 1X PBS for 5 min. Slides were rinsed under running tap water for 1 min prior to staining. Slides were then stained with Hematoxylin 7211 (Richard-Allan Scientific) for 10 min and rinsed under tap water for 1 min and 30 s. Slides were then stained with Eosin-Y alcoholic (Richard-Allan Scientific) for 3 min and rinsed under tap water for 1 min. The slides were then incubated in 100% ethanol for 3 min, allowed to air dry briefly, and mounted with Permount mounting medium (Electron Microscopy Sciences).