The lungs were removed from mice post-mortem, formalin fixed and embedded in paraffin wax before sections of 4 µm were taken and mounted on poly-L-lysine-coated slides (VWR, Leicestershire, UK). Prior to manual staining, sections were de-waxed with xylene (Fisher Scientific, Loughborough, UK) and re-hydrated via graded ethanol: water solutions (Fisher Scientific, Loughborough, UK). Lung sections were stained in accordance with standard histological procedures. H&E sections were evaluated for a range of histopathological changes and assigned a score (0–4 for absent, minimal, mild, moderate and marked, respectively) for each variable, independently assessed by two board-certified veterinary pathologists blinded to the experimental details. The following histopathological variables were assessed semi-quantitatively from each lung section: intra-alveolar haemorrhage, intra-alveolar oedema, intra-alveolar fibrin, alveolar inflammation, alveolar wall necrosis, presence of hyaline membranes, bronchiolar inflammation, type II pneumocyte hyperplasia, vascular necrosis, fibrosis and haemosiderin. In addition, the percentage of tissue affected per lung was recorded. Two sections of lung from different lobes from all mice, in both studies, were subjected to detailed analysis.
Poly l lysine coated slides
Manufactured by Avantor
Sourced in United Kingdom, United States
Poly-l-lysine coated slides are microscope slides with a positively charged coating that enhances cell adhesion. The coating provides a stable surface for the attachment and growth of cells during microscopy and cell-based experiments.
Automatically generated - may contain errors
Lab products found in correlation
Xylene, by Thermo Fisher Scientific (4 mentions)
Paraffin wax, by Avantor (3 mentions)
Neutral buffered formalin, by Merck Group (2 mentions)
1 4 dithioerythritol, by Merck Group (1 mentions)
Papain, by Worthington (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Triton 100, by Merck Group (1 mentions)
Vectashield mounting medium, by Merck Group (1 mentions)
Confocal laser scanning microscopy, by Olympus (1 mentions)
Dylight 488, by Jackson ImmunoResearch (1 mentions)
Cell b software, by Olympus (1 mentions)
Evos cell imaging system, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Cm3050, by Leica (1 mentions)
Biomax mr film, by Kodak (1 mentions)
Formal saline, by Merck Group (1 mentions)
Rnascope 2.5 hd manual dab detection system, by Advanced Cell Diagnostics (1 mentions)
Leitz dmrb fluorescence microscope, by Leica (1 mentions)
13 protocols using poly l lysine coated slides
1
Histopathological Assessment of Murine Lung Samples
2
Isolation and Preservation of Mesenteric Arteries
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Third‐order mesenteric arteries (n = 3 animals as separate experiments) were cleared of attached fat and incubated in HEPES‐buffered physiological solution (NaCl 154 mM, KCl 5.4 mM, MgSO4 1.2 mM, glucose 10 mM, CaCl2 10 mM and HEPES 10 mM; pH 7.4) supplemented with 1,4‐dithioerythritol (Sigma‐Aldrich, Gillingham, UK) at 2 mg/ml and papain (Worthington Biochemical, Lakewood, New Jersey, USA) at 1 mg/ml in a water bath at 37°C for 22 min. The solution was then replaced with HEPES buffer supplemented with type F collagenase from Clostridium histolyticum (Sigma‐Aldrich, Gillingham, UK) at 1 mg/ml for 7 min. The cell suspension was briefly stood on ice before being gently titrated and small drops of cell suspension pipetted onto poly‐l ‐lysine‐coated slides (VWR International, Radnor, Pennsylvania, USA). Slides were left at room temperature for 50 min. before ice‐cold methanol was added to the cell suspension for 10 min. Slides were carefully washed in HEPES buffer before being stored under saline solution at 4°C before being probed for PMCA4 as described below.
3
Immunofluorescence Analysis of Retinal Cell Apoptosis
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For Bcl-2, cleaved caspase-3 (Asp175), and 4′,6-diamidino-2-phenylindole (DAPI) staining 1 and 3 days after ischemia, the eyes were quickly enucleated and dissected, and the posterior eyecups were placed in a chilled fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) for 6 hours. After washing three times, the fixed retinas were transferred into 30% sucrose containing 0.1 M PBS overnight at 4°C and then embedded with Tissue-Tek® O.C.T Compound (Sakura Finetek; Tokyo, Japan) at optimal cutting temperature. Cryostat sections of the retina (12 μm, sagittal) were mounted onto poly-L-lysine-coated slides (VWR International, Radnor, PA, USA). The specimens were blocked in 2% horse serum and 2% BSA in Triton 100 (Sigma-Aldrich) for 60 minutes. The Bcl-2 and cleaved caspase-3 (Asp175) antibodies (1:500) were incubated overnight at 4°C. After three 5-minute rinses in PBS, Dylight488 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) secondary antibody (1:1,000) and DAPI (1:50,000) were added and incubated at room temperature in the dark. After three 5-minute rinses in PBS, the slides were covered with Vectashield Mounting Medium (Sigma-Aldrich). Fluorescence signals were visualized by laser-scanning confocal microscopy (Olympus Corporation, Tokyo, Japan).
4
Tissue Preparation and Immunostaining Protocol
5
Cryosectioning and Fixation of Tumor Tissues
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Was performed at the Advanced Molecular Pathology Lab, IMCB. Both human tumors and mouse tumors formed by human gastric cancer cells (SNU-484) were sectioned into 10 μm slices using a cryostat (Leica) at 16 °C, transferred onto poly-l -lysine-coated slides (VWR), fixed in 4% paraformaldehyde.
6
Histopathological Assessment of Lung Sections
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Four micron thick sections were mounted on poly-l -lysine coated slides (VWR). Prior to manual staining protocols, sections were de-waxed with xylene (Fisher Scientific) and re-hydrated via graded ethanol: water solutions (Fisher Scientific). Lung sections were stained in accordance with standard histological procedures. H&E sections were evaluated for pathological changes associated with disease and assigned a score (0–4; absent, minimal, mild, moderate and marked) for each variable, independently assessed by two board-certified veterinary pathologists blinded to the experimental details.
7
Histological Examination of Lung Samples
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Intact lungs were collected at post-mortem, fixed for 72 h at room temperature in 10% neutral buffered formalin (Sigma-Aldrich, St. Louis, MO, USA) and embedded in paraffin wax (VWR) in accordance with standard histological processes. The samples were examined visually for pathology related markers and scored for severity on a scale of 1 to 4. In addition, four-micron thick sections were mounted on poly-L-lysine coated slides (VWR), de-waxed with xylene (Thermo Fisher Scientific) and re-hydrated via graded ethanol:water solutions (Thermo Fisher Scientific). Hematoxylin and eosin sections were evaluated for pathological changes associated with disease and assigned a score for each variable by two veterinary pathologists, independently. The samples were scored blinded.
8
Ovine Hypothalamic Gene Expression
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The OaTSHβ plasmid (XM_004002368.2) was kindly provided by David Hazlerigg. The OaEya3 plasmid (NM_001161733.1) was cloned as previously described22 (link). The OaCHGA, OaDEC1, OaREVERB-α, CHRONO and OaBMAL2 were cloned as 1,948-1,970 of XM_004017959.4, 407-767 of NM_001129741.1, 1,012-1,411 of NM_001131029.1, 83-532 of XM_027974329.1, and 1,518-1,906 of XM_027965976.1, respectively.
Frozen coronal ovine hypothalamic blocks (n = 4 per group) for in-situ hybridization were cut into 16 µm sections using a cryostat (CM3050s Leica Microsystems, Ltd., Milton Keynes, UK), and thaw mounted onto poly-l-lysine coated slides (VWR International, Lutterworth, UK). Radiolabelled cRNA riboprobes were prepared by plasmid linearization and transcribed using P33 α-UTP (Perkin-Elmer). Fixed sections were hybridized overnight at 60 °C with 5 × 105 cpm of probe per slide. Hybridization signals were visualized on autoradiographic film (Kodak Biomax MR Films, Kodak, USA) after one week exposure at −80 °C. Signal intensity was quantified by densitometry analysis of autoradiographs using the image-Pro Plus 6.0 software (Media Cybernetics, Inc., Marlow, UK).
Frozen coronal ovine hypothalamic blocks (n = 4 per group) for in-situ hybridization were cut into 16 µm sections using a cryostat (CM3050s Leica Microsystems, Ltd., Milton Keynes, UK), and thaw mounted onto poly-l-lysine coated slides (VWR International, Lutterworth, UK). Radiolabelled cRNA riboprobes were prepared by plasmid linearization and transcribed using P33 α-UTP (Perkin-Elmer). Fixed sections were hybridized overnight at 60 °C with 5 × 105 cpm of probe per slide. Hybridization signals were visualized on autoradiographic film (Kodak Biomax MR Films, Kodak, USA) after one week exposure at −80 °C. Signal intensity was quantified by densitometry analysis of autoradiographs using the image-Pro Plus 6.0 software (Media Cybernetics, Inc., Marlow, UK).
9
In situ ZIKV RNA detection in tissues
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Samples covering a range of tissue compartments including main organs, lymphoid tissues, reproductive system, peripheral and central nervous system, including the brain and skin were collected at post-mortem, fixed in 10% formal saline (Sigma) and embedded in paraffin wax (VWR) using previously reported procedures51 (link). Four micron thick sections were mounted on poly-L lysine coated slides (VWR) and prior to treatment de-waxed in xylene and re-hydrated via graded ethanol:water solutions. In situ ZIKV RNA detection was performed using the RNAscope 2.5 HD manual DAB detection system (Advanced Cell Diagnostics, 322300) and a combination of two ZIKV-specific probes (463781 and 464531) in accordance with manufacturer’s instructions. Negative (DaPB 310043) and positive (Hs-PPIB 313901) control probes were used to assess technique efficiency. Equivalent tissue sections from infection naïve RM and tamarins were stained with ZIKV-specific, positive or negative control probes to determine NHP tissue cross-reactivities. Sections were manually counter stained with haematoxylin. RNAscope positive cells were quantified by counting all positive cells within each tissue section and conversion to mean number of positive cells/mm2. Grading definitions were generated using 10 random fields of view (x10 lens and x10 eye-piece magnification; equivalent to an area of 2.2 mm2).
10
Sperm Isolation and Immunocytochemistry
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Semen samples (N, S and P) were thawed and incubated at 37°C for at least 30 min prior to centrifugation on a 60% cushion of Puresperm equilibrated with PureCeption Isotonic Solution (SAGEMedia, UK) mixed with Quinn's Sperm Washing Medium (SAGE, UK) at 400 × g for 20 min. The 60% DGC layers and the pellets were collected and washed twice with Quinn's Sperm Washing Medium at 500xg for 10 min.
For immunocytochemistry, cells recovered from 60% cushions were cytospun at 500 × g for 15 minutes on to poly-l-lysine coated slides (VWR, UK). Cells were fixed in 3:1 methanol/acetone for 15 min, washed with Phosphate Buffered Saline (PBS) and incubated in blocking solution (2% goat serum, 2% serum albumin, 0.1% Triton X-100 in PBS) for 30 min at room temperature (RT). Antibodies were applied to slides in a humidified chamber for 60 min at RT (Supplementary Table 3). Slides were subsequently washed in PBS and incubated for 30 min with appropriate fluorescence-conjugated secondary antibodies in blocking solution at RT. Slides were finally washed in PBS and mounted with an antifade-containing polyvinyl alcohol-mounting medium (DABCO, UK). Fluorescence microscopy was undertaken with a Leica LEITZ DMRB fluorescence microscope. The images were taken with a cooled CCD camera (Orca-03 R, Hamamatsu) controlled by Smart Capture 3 software (DSUK, UK).
For immunocytochemistry, cells recovered from 60% cushions were cytospun at 500 × g for 15 minutes on to poly-l-lysine coated slides (VWR, UK). Cells were fixed in 3:1 methanol/acetone for 15 min, washed with Phosphate Buffered Saline (PBS) and incubated in blocking solution (2% goat serum, 2% serum albumin, 0.1% Triton X-100 in PBS) for 30 min at room temperature (RT). Antibodies were applied to slides in a humidified chamber for 60 min at RT (Supplementary Table 3). Slides were subsequently washed in PBS and incubated for 30 min with appropriate fluorescence-conjugated secondary antibodies in blocking solution at RT. Slides were finally washed in PBS and mounted with an antifade-containing polyvinyl alcohol-mounting medium (DABCO, UK). Fluorescence microscopy was undertaken with a Leica LEITZ DMRB fluorescence microscope. The images were taken with a cooled CCD camera (Orca-03 R, Hamamatsu) controlled by Smart Capture 3 software (DSUK, UK).
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