Ultracut microtome

FTIR spectra were recorded at room temperature at a resolution of 4 cm⁻¹ within the range from 4000 to 400 cm⁻¹ from 32 scans, using a Nicolet 320 FTIR spectrometer and the typical KBr pellet method. NMR spectra were recorded using an INOVA 500 instrument with CDCl₃ or DMSO-d₆ as the solvent. The molecular weights of the DDSQ-based PS block copolymers were evaluated from their ¹H NMR spectra; their polydispersity indices (PDIs) were determined using GPC (Waters 510 apparatus) where the molecular weight calibration was used by PS standard and MALDI-TOF mass spectrometry (Bruker Daltonics Autoflex MALDI-TOF mass spectrometer). The thermal properties of the DDSQ-based block copolymers were determined through DSC using a TA Q-20 instrument; the sample was placed in an aluminum pan and heated at a rate of 20 °C min⁻¹ from room temperature to 250 °C under a N₂ atmosphere (50 mL/min) after the sample was cooled quickly to −90 °C from the first scan. TEM images were recorded using a JEOL 2100 microscope (Japan) operated at 200 kV; ultrathin films were prepared using a Leica Ultracut microtome featuring a diamond knife and placed on a Cu grid coated with a carbon film. The P4VP-b-PS-DDSQ-PS-b-P4VP thin film was imaged after staining with I₂ to display its P4VP segments; the PVPh-b-PS-DDSQ-PS-b-PVPh thin film was stained with RuO₄ to display its PVPh segments.

SH-SY5Y cells were washed and fixed at room temperature (RT) for 1 h in 2.5% glutaraldehyde supplemented with 0.1 M phosphate buffer saline (PBS), and then postfixed in 1.0% osmium tetroxide for 3 h. Next, cells were scraped, spun down, serially dehydrated in ethanol baths, and embedded in blocks of epon Araldite. Ultrathin sections (60–80 nm) were made using an Ultracut Microtome (UC7; Leica), stained with 4% aqueous uranyl acetate and lead citrate for 5 min, and then performed using a TEM (Tecnai G2 20 Twin, FEI) at 200 KV.

Cells were trypsinized, washed with 0.1 M phosphate-buffered saline (PBS) (pH 7.4), and fixed with a solution containing 3% glutaraldehyde/2% paraformaldehyde in 0.1 M PBS (pH 7.4) for 2 h at RT. After fixation, the cells were washed with 0.1 M PBS (pH 7.4) and postfixed with 1% buffered osmium tetroxide for 45 min at RT, and stained with 1% uranyl acetate. After dehydration in graded series ethanol, the cells were embedded in Epon 812 (Fluka) medium and were polymerized at 70 °C for 2 d. Ultrathin sections were cut on a Leica Ultracut microtome and stained with uranyl acetate and lead citrate in a Leica EM Stainer. Digital TEM images were acquired from thin sections using a JEM 1010 transmission electron microscope (JEOL, Peabody, MA) at an accelerating voltage of 80 kV equipped with AMT Imaging System (Advanced Microscopy Techniques, Danvers, MA).

HE staining, IHC, and IF were performed as described previously [19 (link), 20 (link)]. IHC employed the following reagents: anti-Ki67 antibody (Novus Cat. NBP1-40684), anti-SOCS3, and anti-p-Stat3 antibodies (Cell Signaling Technology). IF employed the following reagent: anti-p-Stat3 antibody (Cell Signaling Technology).
For the RNA-FISH assay, the colonic tumor tissues from Apc^+/min mice were recovered at necropsy, washed with PBS, fixed in 4% PFA buffer (paraformaldehyde diluted in PBS that had been pre-treated with Diethylpyrocarbonate). The fixed tissues then were embedded in paraffin. After sectioning with an Ultracut microtome (Leica, Bannockburn, IL, USA), the embedded tissues were dehydrated and digested with Protein K, pre-hybridized for 1 h, and hybridized with miR-708 probe overnight at 37 °C. The miR-708 probe consisted of a 6-carboxyfluorescein (FAM)-labeled oligonucleotide with the sequence 5′-FAM-GGGUCGAUCUAACAUUCGAGGAA-FAM-3′; the oligo was purchased from Guangzhou Ribobio Co., Ltd. (Guangzhou, China). After incubation with 4′,6-diamidino-2-phenylindole(DAPI) staining buffer for 10 min, the sections were mounted and then examined with an Olympus IX81 fluorescent microscope.

Samples were fixed in 1% PFA/2.5% GA in 0.1PB containing 3% sucrose and 1 mM MgSO₄, followed by dehydration in graded methanol and acetone, embedded in Eponate and cut to a 90 nm thickness on a Leica Ultracut microtome. Serial sections were probed using antibodies targeting small molecules L-aspartate, L-glutamate, glycine, L-glutamine, γ-aminobutyric acid (GABA). Antibodies incubated overnight at room temperature were visualized with goat anti-rabbit secondary IgG coated with 1.4 nm gold (Nanoprobes Nanogold® -anti Rabbit IgG) and silver intensified for CMP^{41 (link)}. 8-bit images (243 nm/pixel) were mosaicked and registered with ir-tweak (https://www.sci.utah.edu/download/ncrtoolset.html)^{40 (link)}. RGB channels for display images were linearly contrast stretched for display. Molecular signals were visualized as rgb maps (e.g., γGE → rgb assigns γ-aminobutyric acid, glycine and L-glutamate to red, green, and blue color channels, respectively). For display only, raw data channels were linearly contrast-stretched and sharpened with unsharp masking. Monochrome and RGC images were intensity mapped.