Rnase free pen membrane slides

Sixteen-μm-thick serial frozen sections of rat livers were attached to 2-μm RNase free PEN-membrane slides (Leica, Wetzlar, Germany). Microdissection (Leica, LMD6000) was preceded by a H&E and GSTP staining on serial sections.

Laser-capture microdissection and mRNA isolation were performed as previously reported (Du et al., 2019 (link)). Frozen cerebellar sections (10 μm thick; ∼P100 mice) were cut on a Cryostat NX50 (Leica, Milton Keynes, UK). Sections were attached to RNase free PEN-membrane slides (Leica, Milton Keynes, UK) and stained with fast HE staining (ScyTek Labs, UT, USA). A total of 30 Purkinje cells (somatic and proximal dendritic tissue) were isolated from each side of the cerebellum in three slices per animal, for each group (360 Purkinje cells and six RNA samples per genotype; two mice per group) and collected in the tube using Leica LMD 6500 for subsequent RNA isolation. Total RNAs were extracted using Arcturus Picopure RNA isolation Kit (Thermo Fisher, VA, USA). All samples passed Bioanalyzer RNA QC analysis (Agilent, CA, USA), with a RNA integrity Number (RIN) of at least 7. Each 25 μL reaction solution contained 2 μL primers, 12.5 μL SYBR green Supermix (BioRad, CA, USA) and was run under the following conditions: 50°C 2 min; 95°C 10 min; 95°C 15 s, 60°C 1 min for 40 cycles, followed by a melting curve. As a relative quantification, fold changes were measured using the ΔΔCt method, with Gapdh as an internal control.