The 5-μm-thick sections were fixed with 10% formalin followed by hydration in water. The tissue was stained with periodic acid solution (Sigma) for 5 min, Schiff's reagent for 15 min, and hematoxylin solution for 90 sec. Samples were dehydrated in increasing concentrations of ethanol (70%, 90%, 95%, and 100%) followed by incubation in xylene and finally sealed with a coverslip using Fisher Chemical™ Permount™ Mounting Medium (Fisher Scientific). Goblet cells were observed and counted in individual well-preserved colonic crypts using a Leica DMRBE microscope.
Fisher chemical permount mounting medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, Ireland
Fisher Chemical™ Permount™ Mounting Medium is a xylene-based mounting medium used for permanently mounting specimens on microscope slides. It is designed to provide a clear, durable, and long-lasting seal for microscopic preparations.
Automatically generated - may contain errors
Lab products found in correlation
Periodic acid solution, by Merck Group (1 mentions)
Dmrbe microscope, by Leica camera (1 mentions)
Bond polymer refine detection, by Leica Biosystems (1 mentions)
Leica bond, by Leica camera (1 mentions)
Signalstain antibody diluent, by Cell Signaling Technology (1 mentions)
Halo platform, by Indica Labs (1 mentions)
Hematoxylin, by Abcam (1 mentions)
Karyomax colcemid solution, by Thermo Fisher Scientific (1 mentions)
Rm2235, by Leica camera (1 mentions)
Dm1000, by Leica camera (1 mentions)
Formic acid, by Merck Group (1 mentions)
5 protocols using fisher chemical permount mounting medium
1
Quantification of Colonic Goblet Cells
2
Immunohistochemical Analysis of β-catenin
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FFPE slides were treated sequentially following the Leica BOND RX default Bake & Dewax Protocol, HIER (ER2, 20 min, 95°C) protocol and the immunohistochemistry (IHC) Leica BOND protocol (anti-β-catenin antibody, Cell Signaling Technology, clone: 15B8, cat. 37 447S; SignalStain Antibody Diluent, Cell Signaling Technology, cat. 8112; BOND Polymer Refine Detection, Leica Biosystems, cat. DS9800). Stained slides were treated with 95% ethanol for 10 s twice and 100% ethanol for 10 s twice, followed by xylene for 10 s twice. Slides were mounted with Fisher Chemical Permount Mounting Medium (Fisher Scientific, cat. SP15-100) and scanned on HAMAMATSU NanoZoomer 2.0RS. Images were analyzed using the HALO platform (Indica Labs).
3
Quantification of Osteoclast Activity
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Paraffin-embedded joint slides were left on a hot plate at 55° C for 10 mins, deparaffinized by xylene, and hydrated through a graded ethanol series. The sections were stained for TRAP-positive cells using the K-assay Trap staining kit (Kamiya-Biomedical Company, #KT-008, USA) per the manufacturer’s instruction. After staining, sections were washed by distilled water, stained with hematoxylin (Abcam, USA), dehydrated through ascending ethanol grades, submerged in xylene for 1 min, and mounted with Fisher Chemical™ Permount™ Mounting Medium (Fisher, #SP15-100, USA). Bone marrow images in the subchondral bone were obtained by light microscopy (Olympus, DP71). The percentage of the TRAP-positive area was then measured using Image J (NIH, USA). For quantification, the percentage of the positively stained area was divided by the total subchondral bone marrow area using Image J (NIH, USA).
4
Chromosomal Spread Preparation and Analysis
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Metaphase chromosome spreads were prepared by incubating cells with 100 μg/ml KaryoMAX Colcemid Solution in PBS (Gibco) for 3 hr. Cells were harvested at 1000 rpm and resuspended in 75 mM KCl at 37°C for 15 min. Cells were fixed in a 3:1 mixture of ice-cold methanol/acetic acid at least overnight at –20°C. Samples were then dropped onto pre-cleaned slides, briefly steamed (<5 s) over an 80°C water bath to disperse nuclei and air-dried overnight at room temperature. Slides were stained in Giemsa solution and mounted using Fisher Chemical Permount Mounting Medium (Thermo Fisher Scientific). Images were acquired on an Olympus IX50-S8F microscope using a 100× objective and images were analyzed using ImageJ.
5
Histological Evaluation of Dental Pulp Exposure
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After fixation, the samples were rinsed in phosphatebuffered saline for 20 min and then decalcified with 20% formic acid (Sigma Aldrich), which was stirred at room temperature for 3-4 weeks. The samples were then dehydrated in increasing concentrations of ethanol, cleaned, and embedded in paraffin. The paraffin-embedded teeth were sagittally sectioned at a thickness of about 3 μm with a microtome (RM2235; Leica, São Paulo, SP, Brazil). The paraffin sections were stained with hematoxylin and eosin (HE) and mounted with Fisher Chemical™ Permount™ Mounting Medium (Fischer Scientific, Ireland Ltd., Dublin, LE, Ireland).
A previously calibrated experienced pathologist who was blinded to sample group assignment performed histological evaluation of the HE-stained sections based on the ISO 7405-2008 (19, 20) .
All sections (three per slide) were viewed under an optical microscope (DM1000, Leica, São Paulo, SP, Brazil). The pathologist performed descriptive analysis and assigned scores for inflammatory response and tissue organization adjacent to the pulp exposure (Table 1).
In order to test the intra-examiner variability, Kappa coefficient was obtained using a sample of 10 sections. The Kappa value for intra-examiner variability was 0.91, showing excellent agreement.
A previously calibrated experienced pathologist who was blinded to sample group assignment performed histological evaluation of the HE-stained sections based on the ISO 7405-2008 (19, 20) .
All sections (three per slide) were viewed under an optical microscope (DM1000, Leica, São Paulo, SP, Brazil). The pathologist performed descriptive analysis and assigned scores for inflammatory response and tissue organization adjacent to the pulp exposure (Table 1).
In order to test the intra-examiner variability, Kappa coefficient was obtained using a sample of 10 sections. The Kappa value for intra-examiner variability was 0.91, showing excellent agreement.
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