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Superfrost ultra plus adhesion slides

Manufactured by Thermo Fisher Scientific
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Superfrost Ultra Plus Adhesion Slides are microscope slides designed to provide a superior surface for tissue and cell attachment. These slides feature a unique coating that enhances the adherence of biological samples, ensuring secure specimen mounting and improved slide performance.

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14 protocols using superfrost ultra plus adhesion slides

1

Immunofluorescence Imaging of 3D NSC Spheroids

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3D NSC spheroids were fixed with 4% PFA in 0.1 mol/L phosphate buffer for 1 h at RT and subsequently washed 3 times with phosphate buffered saline (PBS). Spheroids were permeabilized with 0.2% TritonX-100 in PBS and blocked in 3% BSA in PBS at RT for 1 h. Spheroids were incubated with primary antibodies (Supplementary Table S1) overnight at 4°C. Spheroids were then washed 3 times in PBS before their incubation with isotype-specific secondary antibodies diluted in 3% BSA in PBS for 1 h at RT. Spheroids were washed 3 times in PBS and mounted on Superfrost™ Ultra Plus Adhesion Slides (Thermo Fisher Scientific) using ProLong™ Diamond Antifade Mountant with DAPI for nuclear labelling. Image acquisition was undertaken using a BX-41 epifluorescent microscope (objectives: ×20 0.50 NA; 40 × 0.75 NA; Olympus) fitted with a DP-74 digital camera and Cellsens software (V1.18; Olympus).
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2

Mouse Brain Tissue Fixation and Sectioning

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Brain tissues from 2-wk-old mice were dissected out, immersed immediately in ice-cold fixative (4% formaldehyde in 0.1 M phosphate buffer) and kept in the same fixative overnight at 4 °C. Brains were then transferred to increasing concentrations of sucrose (10%, 20% and 30% wt/vol) in PBS, embedded in Tissue-Tek OCT compound, and frozen in liquid nitrogen-cooled isopentane. Coronal or sagittal (15 to 30 μm thick) sections were cut with a cryostat and mounted on Superfrost Ultra Plus Adhesion slides (Thermo Fisher Scientific). Sections were then blocked and permeabilized with a solution containing 3% normal goat serum, 1% BSA, PBS, and 0.1% Triton-X100 for 1 h at room temperature; incubated with primary antibodies (diluted in the same buffer) overnight at 4 °C; washed; incubated with Alexa Fluor-conjugated secondary antibodies for 1 h at room temperature; and finally mounted with Prolong Gold antifade reagent with DAPI and sealed with nail polish. Images were acquired with a PerkinElmer Ultraview spinning disk confocal microscope equipped with a 40× objective.
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3

Immunohistochemical Analysis of Fibronectin, HNE, and NT

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Paraffin sections on Superfrost Ultra Plus Adhesion Slides (Thermo Fisher Scientific Inc, Waltham, MA, USA) were deparaffinized and rehydrated in ethanol. Fibronectin immunohistochemistry was performed with rabbit polyclonal anti-fibronectin antibody (1:2000, Sigma-Aldrich, Budapest, Hungary), using the avidin–biotin method [30 (link)]. HNE and NT immunohistochemistry was performed with mouse monoclonal antibody (HNE clone: HNEJ-2, JaICA, Japan; NT clone: #189542, Cayman Chemical Company, Michigan, IL). Color development was induced by incubation with diaminobenzidine (DAB) kit (Vector Laboratories, Burlingame, CA). Pictures were taken from the stained sections for further analysis. The fibronectin stained area was quantified with Image J software.
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4

Immunostaining of Phosphorylated RNAPII

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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.
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5

Perfusion and Sectioning for Mouse Brain and Olfactory Epithelium

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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.
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6

Tissue Harvesting and Preservation

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For tissue collection, mice were killed by cervical dislocation. TA from mice were collected and frozen in liquid nitrogen-cooled isopentane for sectioning or in liquid nitrogen for total RNA isolation. For histological analysis, tissue specimens were sliced using a cryostat microtome (CM1510S, Leica) to 8 μm thick and mounted onto Thermo Scientific SuperFrost Ultra Plus Adhesion Slides.
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7

Precise Brain Tissue Harvesting and Preservation

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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.
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8

Histological Analysis of Tissue Samples

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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).
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9

Quantitative Histological Analysis of Kidney Fibrosis

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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.
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10

Immunohistochemical Analysis of GluN1 and PSY+ IgG in Mouse Hippocampus

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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 (αGluN1N-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).
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