Spleen tissues from 4 month-old mice (Pten+/+, PtenG129E/+ and PtenC124S/+ mice) were dissected and fixed in 4% paraformaldehyde for IHC analysis. For staining, tissues were fixed in 4% paraformaldehyde overnight, paraffin embedded, and then sectioned at 5 μm. After deparaffinization and rehydration, antigen retrieval was performed in a pressure cooker with sodium citrate buffer at 95°C for 25 minutes. Sections were incubated in a 0.3% H2O2 solution in 1× PBS, and then a 10% serum solution in 1 × PBS for 30 minutes each solution was used to block endogenous peroxidase and background from the secondary antibody, respectively. The sections were stained with the γH2ax (Cell Signaling #9718, 1:500) in 1 × PBS at 4°C overnight, and incubated in a biotinylated anti-rabbit secondary antibody in 1 × PBS (1:1000) at room temperature for 30 minutes. The Vectastain ABC Elite kit was used to enhance specific staining, and the staining was visualized using a 3’-diaminobenzidine (DAB) substrate. Stained sections were counterstained using hematoxylin and dehydrated before they were sealed with a coverslip with Richard-Allan Scientific® Cytoseal™ XYL Mounting Medium. Stained slides were visualized by a bright-field microscope.
Cytoseal xyl mounting medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Cytoseal XYL is a water-soluble, xylene-based mounting medium for use in microscopy applications. It is designed to provide a durable, long-lasting mount for tissue sections and other specimens.
Automatically generated - may contain errors
Lab products found in correlation
Axioplan 2 imaging microscope, by Zeiss (2 mentions)
Poly l lysine solution, by Merck Group (2 mentions)
Achroplan 63 0.9w dic 3 objective, by Zeiss (2 mentions)
Bx40 microscope, by Olympus (2 mentions)
Rm2255 microtome, by Leica camera (2 mentions)
Tissuegnostic histological microscope, by Zeiss (2 mentions)
γh2ax, by Cell Signaling Technology (1 mentions)
Histochoice, by Merck Group (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
Ab150686, by Abcam (1 mentions)
Nis elements software, by Nikon (1 mentions)
Superfrost plus, by Avantor (1 mentions)
H e staining kit, by Abcam (1 mentions)
Sucrose, by Merck Group (1 mentions)
Bz x710, by Keyence (1 mentions)
Cryostat, by Leica Biosystems (1 mentions)
Envision system hrp labelled polymer secondary antibody, by Agilent Technologies (1 mentions)
Liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
Anti il 6 antibody, by Abcam (1 mentions)
Dab chromogen system, by Vector Laboratories (1 mentions)
12 protocols using cytoseal xyl mounting medium
1
Immunohistochemical Analysis of Pten Mutant Mouse Spleen
2
Tissue Sectioning and Slide Preparation
3
Visualizing DNA Repair Foci in Living Cells
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The cells were prepared as described in spot assay. For visualizing DNA in living cells, 10 mg/ml DAPI was added to the culture 30 min before imaging. Next, 1 ml aliquots were washed, pelleted and resuspended in 200μl of fresh synthetic minimal medium (SD). Then 5 ml volume of cells were immobilized on a glass slide precoated with 0.1% poly-L-lysine solutions (Sigma), to prevent evaporation, the cover glass was sealed with Cytoseal™ XYL mounting medium (Richard-Allan Scientific). Cells were viewed under a Zeiss Axioplan 2 imaging microscope (Carl Zeiss, Thornwood, NY) with a water-immersion Achroplan 63_/0.9W/DIC III objective. The illumination source was a 100-W mercury arc lamp. Cell images were taken using an AxioCamHRm digital camera operated via AxioVision 4.5 software. Confocal images were captured with a LSM 510 META system operated via META 3.2 software. The wavelengths of the filters used to visualize the RAD52-GFP (excitation 488 nm; emission 509 nm) and 4',6-diamidino-2-phenylindole (DAPI; excitation 358 nm, emission 463 nm). Image acquisition times for RAD52-GFP was 1100 ms. At least 300 nuclei were counted for total of 6 – 10 field of cells, the observations of RAD52-GFP foci formation are from 1.5 to 6 hr after MNNG treatment, the results shown are based on 3 independent experiments. Unless otherwise noted, all experiments were performed at room temperature.
4
Visualizing DNA Repair Foci in Living Cells
5
Histological Evaluation of Bone Fusion
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After micro‐CT imaging, representative spines from each group underwent histological evaluation for visualization of the fusion bed. Samples were demineralized as described elsewhere and embedded in paraffin.20 Seven‐micrometer thin sections were cut with a RM2255 microtome (Leica), deparaffinized, and stained with Gill hematoxylin and eosin (Sigma‐Aldrich) according to the manufacturer's recommendations. Stained sections were mounted with Cytoseal XYL mounting medium (Thermo Scientific), imaged on a TissueGnostic histological microscope (Zeiss), and visualized using TissueFAXS software.
6
Histological Analysis of Grafts
7
Histological Analysis of Intracerebral Hemorrhage
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Brains were removed from perfusion-fixed mice and stored overnight in 4% PFA. Brains were then serially dehydrated in 7.5%, 15%, and 30% sucrose (Sigma-Aldrich) solutions at 4°C and frozen in OCT sectioning medium at −80°C. The tissue was sectioned with a cryostat (Leica Biosystems, Wetzlar, Germany), generating 35 µm thick sections collected every 500 µm. Sections were mounted on glass slides (Superfrost Plus, VWR International, Radnor, PA) and refrigerated overnight. These sections were then stained with hematoxylin and eosin using the H&E staining kit (Abcam, Cambridge, United Kingdom), per the manufacturer’s instructions. Stained sections were sealed using the Cytoseal XYL mounting medium (Thermo Fisher Scientific) and allowed to dry for a minimum of 24 h. Sections were imaged using a bright field microscope (BZ-X710, Keyence Corporation, Osaka, Japan) at ×4 magnification.
Intracerebral hemorrhage lesions were identified as an abnormal appearance of blood within the brain tissue (appears red by eosin staining). Serial coronal sections of brains were viewed by light microscopy, and the number of hemorrhagic lesions within each of the following brain regions was recorded: cerebral cortex, basal ganglia, midbrain, brainstem, or cerebellum. ImageJ image analysis software was used to measure each lesion’s maximum area (mm2).
Intracerebral hemorrhage lesions were identified as an abnormal appearance of blood within the brain tissue (appears red by eosin staining). Serial coronal sections of brains were viewed by light microscopy, and the number of hemorrhagic lesions within each of the following brain regions was recorded: cerebral cortex, basal ganglia, midbrain, brainstem, or cerebellum. ImageJ image analysis software was used to measure each lesion’s maximum area (mm2).
8
Histological Analysis of Implant Samples
9
Immunohistochemical Analysis of Ovarian Cancer
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For morphological and immunohistochemical analysis of OC tissue, paraffin‐embedded ovarian abdominal tumours 10 μm thick sections were generated. Deparaffinization of the slides was performed through a series of incubations in xylene, 100% ethanol, 95% ethanol, 70% ethanol, and deionized water. Antigen retrieval was performed by treatment in 10 mM citrate buffer solution for 30 min at sub‐boiling temperature. Endogenous peroxidase activity was inhibited by a 10 min incubation in 3% H2O2. Sections were subsequently blocked in Tris‐buffered saline (TBS)‐Tween buffer containing 5% goat serum for 1 h at room temperature. A primary rabbit anti‐IL‐6 antibody (#208113; Abcam, Cambridge, MA, USA) was added (1:50) and incubated overnight in a humidified box at 4°C. Normal rabbit IgG were used as negative controls. Sections were then incubated with the ‘EnVision+System‐HRP‐labelled polymer secondary antibody’ (DAKO, Carpinteria, CA, USA) for 1 h at room temperature. The reaction was then visualized with the DAKO ‘Liquid DAB+substrate chromogen system’ according to the manufacturer's instructions. Slides were counterstained with Harrys Haematoxylin (1:1 in deionized water) for up to 5 min, dehydrated with incubations in 70% ethanol, 95% ethanol, 100% ethanol, and xylene, and mounted with xylene‐based Cytoseal XYL mounting medium (Thermo Fisher Scientific, Suwanee, GA, USA).
10
Mutant SOD1 Expression in Muscle Sections
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Immunohistochemistry of mice muscle sections (Wt and G93A*SOD1) for SOD1 was used to observe the expression pattern of mutant-SOD1 in muscle sections, as described previously [55 (link)]. Briefly, d thin serial sections of 5 μm from paraffin-embedded tissues were subjected to subsequent deparaffinization, hydration, antigen retrieval (H-3300, Vector Laboratories, Burlingame, CA, USA), blocking of endogenous peroxidases (0.3% v/v hydrogen peroxide; Bloxall, SP-6000, Vector Laboratories, Burlingame, CA, USA), blocking with 5% serum (Vector Laboratories, Burlingame, CA, USA), and primary antibody incubation in blocking serum overnight. The sections were incubated with secondary antibody (Vector Laboratories, Burlingame, CA, USA) amplified antigen-antibody interaction using VECTASTAIN Elite ABC Peroxidase kit (PK-6100, Vector Laboratories, Burlingame, CA, USA) and visualized using DAB-chromogen System (SK-4105, Vector Laboratories, Burlingame, CA, USA). Subsequently, the sections were counterstained with hematoxylin, mounted using Cytoseal XYL mounting medium (Thermo Scientific), and imaged using Olympus BX40 microscope (Olympus Life Science, Waltham, MA, USA). The primary antibody used in this experiment is SOD1 (1:100; 37385; Cell Signaling).
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