Sm2010r sliding microtome

Free floating 45 μm thick sagittal sections were cut using a Leica SM2010 R sliding microtome and transferred to sterile TBS for storage. Sections were gathered and placed sequentially into wells (∼4 per well). Sections were then randomly selected from each well to perform antibody staining using the primary antibodies ACU193 (0.2 μg/ml), Alexa Fluor^® 555-conjugated NU4 (0.92 μg/ml), Cy3-conjugated anti-GFAP (1:800, Sigma) and the secondary antibody Alexa Fluor^® 633 goat anti-human IgG (1:2000, Invitrogen). Floating slices were rinsed 3 × 10 min with TBS and blocked with blocking buffer (10% NGS with 0.3% Triton X-100 in TBS) for 60 min at RT. Slices were then incubated with the respective antibodies in blocking buffer overnight at 4°C with gentle rotation. Sections were washed 3 × 10 min in TBS and incubated with secondary antibody for 3 h at RT with orbital agitation in the dark. Secondary was prepared in blocking buffer diluted 10-fold with TBS. Sections were then washed 3 × 10 min in TBS, mounted using ProLong Diamond^® antifade mounting media with DAPI (Invitrogen) and 24 × 60 mm No.1.5 glass coverslips (Thermo Scientific). Z-stacks of the brain sections were collected at 10× or 100× on a Leica SP5 confocal microscope and analyzed with ImageJ.

The plant material, SP80–3280 (control) and the L6 ShF5H1 transgenic line (13 months old), was cultivated by Nuseed Brasil company in field trials located at Barra de São Miguel, Alagoas, with coordinates 9.817666°S (latitude) and 35.956677°W (longitude). This area was designated for Planned Release of Material into the Environment. The 8^th internodes of the culms were separated and sent to the Plant Anatomy Laboratory of the Department of Plant Biology at the Institute of Biology of the State University of Campinas, Campinas-SP. Samples from the mid-region of the 8^th internode of the culm were fixed in FAA 50 for 48 hours⁷³ and subsequently stored in 70% ethanol solution. For light microscopy analyses, each sample was sectioned using a Leica SM 2010 R sliding microtome with a thickness of 18–20 µm. Subsequently, the obtained sections were clarified with sodium hypochlorite for 5 minutes, underwent distilled water baths, and were stained with Alcian Blue for 30 seconds.⁷³ Portions of the sections were subjected to the following histochemical tests for lignin detection: Mäule reagent and Phloroglucinol-HLC. All sections were mounted between a slide and coverslip, with result documentation carried out by capturing images using an Olympus DP71 video camera attached to an Olympus B× 51microscope.