Superfrost ultra plus adhesion slides

Slices measuring 3–5 µm were cut from paraffin-embedded, formalin-fixed liver pieces. Slices were mounted on microscope slides (Superfrost Ultra Plus Adhesion Slides, Thermo Fisher Scientific). For RNAPII staining, samples were deparaffinized with xylene, rehydrated with an alcohol gradient and washed with Milli-Q water before antigen retrieval (30 min in citrate buffer, pH 6). The antibodies used were: Alexa Fluor 594-RPB1 antibody (in 1:250 dilution), recognizing all forms of RNAPII independently of the phosphorylation status of their CTD (cat. no. 664908, BioLegend); RNAPII-ser2p (cat. no. ab5095, Abcam); RNAPII-ser5p (cat. no. ab5131, Abcam); phospho-ATM (Ser1981, cat. no. 4526, Cell Signaling Technology); and phospho-histone H2A.X (Ser139, cat. no. 9718; Cell Signaling Technology) all in 1:500 dilution. To reduce the background fluorescence level, a mouse-on-mouse detection kit was used (cat. no. BMK-2202, Vector Laboratories). Sections were counterstained using DAPI or Hoechst 33342.

The intact C57BL/6 male mice were deeply anaesthetised by intraperitoneal urethane injection (2.4 g/kg) and transcardially perfused with 20 mL of ice-cold 0.1 M phosphate-buffered saline (PBS, pH: 7.4) followed by 150 mL 4% paraformaldehyde (PFA) solution in Millonig buffer (pH 7.4) for 15 min. After perfusion, the brains with OBs were removed, and collected into PFA for 36 h for postfixation at 4 °C. Olfactory epithelia were also removed and collected into 4% PFA containing 30% sucrose for 36 h postfixation, to ensure cryoprotection. The brains with the OBs were coronally sectioned using a Leica VT1000 S vibratome (Leica Biosystems, Wetzlar, Germany). Five series of 30 µm sections were gathered and stored in antifreeze solution (20% ethylene glycol, 30% glycerol and 0.1 M sodium-phosphate buffer) at −20 °C. OE samples were embedded in OCT tissue freezing medium (Leica), and five series of 17 µm transversal sections were obtained using a cryostat (Leica CM1950, Nussloch, Germany). Then, sections were taken on SuperFrost Ultra Plus adhesion slides (Thermo Fisher Scientific, Braunschweig, Germany, Cat. No.: 10417002), and stored at −20 °C.

Mice were swiftly killed by cervical dislocation, brains were removed and instantly flash frozen with liquid nitrogen and stored in −80°C. Brain sections and tissue punches were conducted in a Leica CM 1950 cryostat. Samples for qPCR were dissected from 1-mm-thick coronal slices (from bregma: AP 1.18 mm, ML ±3 mm, DV 3.6 mm, to bregma: AP 0.14 mm, ML ±3.6 mm, DV 4.1 mm, using Franklin and Paxinos, 2007 ) with a 1-mm diameter tissue punching device. Samples for RNAscope were sliced in 20-μm sections, from bregma AP 1.25–0.25 mm, mounted directly onto SuperFrost Ultra Plus Adhesion slides (Thermo Fisher Scientific) and kept in −80°C.

After collection, tissues were fixed in 10% neutral buffered formalin (Sigma) for 48 h followed by processing for paraffin embedding (HistoStar Embedding Workstation). Sections of 7 µm thickness from the paraffin-embedded tissues (Thermo Scientific Microm HM355S microtome) were mounted on Superfrost Ultra Plus Adhesion slides (Thermo Scientific) and routinely stained with hematoxylin and eosin (Mayers Haematoxylin 1 l, 3801582E, Leica; Eosin Y solution, aqueous (1 L), HT110232-1L, Sigma-Aldrich) for histopathological examination. Then, sections were stained with periodic acid–Schiff (PAS). Slides were incubated in freshly prepared periodic acid (0.5%) for 15 min, rinsed, and incubated for 5 min in distilled water. The Schiff reagent (3952016-500ML, Sigma-Aldrich) was added onto the slides and kept for 15 min at room temperature in dark. The sections were then washed in slightly lukewarm running tap water for 5 min, rinsed and incubated in distilled water for 2 min, and counterstained in Mayer’s hematoxylin for 1 min. After washing in running tap water for 5 min, sections were dehydrated (95% EtOH, 100% EtOH, 100% EtOH, 3 min each, followed by two times xylene for 5 min) and mounted in DPX mounting medium (06,522, Sigma).

Paraffin sections mounted on Superfrost Ultra Plus Adhesion Slides (Thermo Fisher Scientific Inc, Waltham, MA, USA) were deparaffinized and rehydrated in ethanol. Desmin, αSMA and fibronectin immunohistochemistry was performed with polyclonal antibodies (anti-desmin MS 376-S1, Thermo Fisher, 1:1000; anti- αSMA ab5694, Abcam, 1:1000; anti-FN HPA0027066, Atlas Antibodies, 1:1000), using the avidin–biotin method and developed by incubation with diaminobenzidine (Vector Laboratories, Burlingame, CA, USA). Pictures were taken with Zeiss AxioCam 512 of the stained sections for further analysis. Glomerular tuft was delineated manually and standard glomerulus size parameters (e.g., Feret diameter) as wells as the PAS, desmin, and αSMA positive area were determined using the CellProfiler cell image analysis software.
Lipid deposition was determined by oil-Red-O (O0625, Sigma-Aldrich, Budapest, Hungary) staining of 5-μm-thick cryosections.

Mice (P22) hippocampal coronal sections of 50 μm were incubated overnight at 4 °C with either a polyclonal antibody against the N-terminal part of the GluN1 subunit (αGluN1_N-term Alomone Labs, 20 μg ml⁻¹) or human purified IgG (20 μg ml⁻¹). Fluorescent revelation was carried out with secondary anti-rabbit or anti-human Alexa 488 antibodies (Life Technologies, 1/1,000) for 2 h at room temperature. Images were obtained using a Nanozoomer and a confocal microscope (SP8, Leica). Brains (provided by A. Ramsey) from wild-type and GluN1-knockdown animals at 14 weeks were perfused and stored at − 20 °C. Coronal tissue sections of hippocampal areas (20 µm thick) were cut on a microtome-cryostat, thaw-mounted onto Thermo Scientific, SuperFrost Ultra Plus adhesion slides and stored at − 20 °C until further processing. Sections were fixed at 4 °C in 4% paraformaldehyde. Blocking was carried out in a 1 × TBS solution in 0.3 M glycine containing 10% normal goat serum (Sigma) and incubation with PSY + purified IgG (5 μg ml⁻¹, pooled from two different patients) was done in a 1 × TBS solution containing 10% normal goat serum overnight at 4 °C. Staining with secondary antibody anti-human Alexa 568 (Invitrogen, 1/500) was performed for 1 h and slides were mounted with Vectashield + Dapi (Vector Laboratories). Image acquisition was done on a video spinning-disk system (Leica DMI6000B, × 40).