Capsure lcm macro caps

Fifteen-micron-thick cryostat sections were mounted onto non-polarized glass slides (Gold Seal Rite-On Ultra Frost, Thermo-Scientific), thawed, slightly fixed in 75% ethanol for 40 sec, and stained with Thioflavin-S 0.05% in 100% ethanol (ThioS, Sigma-Aldrich, Cat# 1326-12-1) for 5 min, followed by dehydration in increasing concentrations of ethanol and xylene, and air dried. Immediately, sections were placed onto the stage of an Arcturus Veritas LCM apparatus (Arcturus, Thermo-Scientific) and laser capture microdissection (LCM) was performed with a laser power of 80–85 μV and pulse duration of 3,500 μsec to fill up ten CapSure LCM Macro caps (Thermo-Scientific, Cat# LCM0211) per region of interest and per donor. In CTRL donors, approximately 100 mm² of cortex were dissected. In AD tissues, four different regions of interest were dissected: ThioS+ plaques (approx. 1,000/donor), the 50 μm area around those ThioS+ plaques, ThioS+ NFTs with the 50 μm area around them (approx. 1,500/donor), and cortex far (>50 μm) from both ThioS+ plaques and NFTs (approx. 80–100 mm²).

Kidneys from 3 naïve adult male SD rats, 6 months old, were collected at necropsy into optimum cutting temperature embedding medium, frozen on dry ice and isopentane, and stored at ≤ −70°C until use. Frozen blocks containing kidney tissue were sectioned at 8 µm and adhered to glass slides. Serial sections were stained with rabbit α-glutathione-s-transferase, sheep anti-Tamm-Horsfall protein, or rabbit anti-aquaporin-2 (all from EMD Millipore) to identify proximal tubules, the TAL of the loop of Henle, and CD, respectively (Bauchet et al., 2011 (link)) using the HistoGene LCM immunofluorescence staining kit (Thermo Fisher Scientific) and Alexa Fluor 488-conjugated donkey anti-rabbit or donkey anti-sheep secondary antibodies (Thermo Fisher Scientific) as appropriate. Additional serial sections were stained with a histochemical stain (HistoGene LCM frozen section staining kit; Thermo Fisher Scientific) to identify glomeruli. Nephron segments of interest were identified under fluorescence or light microscopy as appropriate and collected from the stained slides using an Arcturus XT LCM instrument onto CapSure LCM Macro caps (Thermo Fisher Scientific). For samples used for sRNA-seq and ddPCR, RNA was isolated using the miRNeasy Micro kit (Qiagen). For samples used in the FirePlex analysis, RNA isolation was not performed.

Fifteen-micron-thick cryostat sections were mounted onto non-polarized glass slides (Gold Seal Rite-On Ultra Frost, Thermo-Scientific), thawed, slightly fixed in 75% ethanol for 40 s, and stained with Thioflavin-S 0.05% in 100% ethanol (ThioS, Sigma-Aldrich, Cat# 1326-12-1) for 5 min, followed by dehydration in increasing concentrations of ethanol and xylene, and air dried. Immediately, sections were placed onto the stage of an Arcturus Veritas LCM apparatus (Arcturus, Thermo-Scientific) and laser capture microdissection (LCM) was performed with a laser power of 80–85 μV and pulse duration of 3500 μs to fill up 10 CapSure LCM Macro caps (Thermo-Scientific, Cat# LCM0211) per region of interest and per donor. In CTRL donors, approximately 100 mm² of cortex were dissected. In AD tissues, four different regions of interest were dissected: ThioS+ plaques (approximately 1000/donor), the 50 μm area around those ThioS+ plaques, ThioS+ NFTs with the 50 μm area around them (approximately 1500/donor), and cortex far (> 50 μm) from both ThioS+ plaques and NFTs (approximately 80–100 mm²).