Pen slides

All procedures were performed in an RNase-free environment. Animals were sacrificed under deep CO₂-induced anesthesia, brains were quickly dissected in ice-cold PBS, cryopreserved in 20% sucrose overnight, frozen in OCT compound (Polysciences), and stored at −80 °C. Twenty μm thick coronal sections were collected on UV-treated and 0.05% poly-L-lysine coated membrane-covered PEN slides (Zeiss), fixed for 1 min in ice-cold 70% EtOH, incubated for 45 sec in 1% cresyl violet acetate solution (Waldeck) and washed for 1 min each in 70% EtOH and 100% EtOH. Sections were briefly dried on a 37 °C warming plate and immediately processed. The granule cell layer of the DG was isolated by laser capture microdissection using a PALM MicroBeam Rel.4.2 (Zeiss). RNA was isolated from the collected tissue using Rneasy Micro Kit (Qiagen) and reverse transcribed using the SensiFast cDNA Synthesis Kit (Bioline). Quantitative real-time PCR was performed in triplets for each sample using the LightCycler DNA Master SYBR Green I Kit in a LightCycler 480 System (Roche). The relative copy number of Gapdh RNA was used for normalization. Data were analyzed using the comparative CT method (Schmittgen and Livak, 2008 (link)).

Freshly harvested embryos were frozen in the OCT, sectioned at 10μm thickness and transferred to PEN slides (Zeiss). Slides were dipped in 95% ethanol for 2 min to fix the samples and stained with crystal violet stain (3% in ethanol) on ice. This was followed by dipping in 70% ethanol for 30–40 sec to remove the OCT and dehydration in 100% ethanol for 2 min. The periocular mesenchymal tissue was micro-dissected using Laser capture microscope (Zeiss AxioObserver.Z1 inverted microscope). 500 pg of RNA was isolated from each sample, converted to cDNA and amplified using Nugen Ovation kit (Nugen) to obtain 2–3 μg cDNA, which was then converted to cDNA library for RNA-sequencing analysis at core facility in Columbia University. The RNAseq data is available at the GEO repository under accession number GSE103402.

Patient frozen LN tissue from the pathology department of the Amsterdam UMC hospital was used in this study. (LN studied was a cervical LN resected out of a 46-yr-old woman suffering from chronic sialadenitis. Patient material was obtained according to the ethical standards of our institutional medical ethical committee, as well as in agreement with the 1975 Declaration of Helsinki as revised in 1983). The LN was fresh-frozen in liquid nitrogen shortly after surgical resection. Five serial cryosections (10 μm per cryosection, CryoStar NX-70; Thermo Fisher Scientific) of the LN tissue on PEN slides (1 mm; Zeiss) were used to obtain higher DNA concentrations. Immunostaining was performed to visualize the GCs. The PEN slides were incubated with drops of hematoxylin (KLINPATH; VWR Life Science) for 1 min at RT, after a quick and gentle wash with demi water and tap water, they dried overnight at RT. In parallel, H&E staining of the frozen tissue was performed on normal (TOMO) slides at Amsterdam UMC diagnostics.