Frozen hippocampal sections were stained with the PAS method according to the standard procedure described previously (Augé et al., 2017 (link)). Briefly, sections were fixed for 10 min in Carnoy’s solution (60% ethanol, 30% chloroform and 10% glacial acetic acid). They were then pre-treated for 10 min with 0.25% periodic acid (19324–50, Electron Microscopy Sciences) in distilled water, followed by a washing step for 3 min with distilled water. Samples were immersed in Schiff’s reagent (26052–06, Electron Microscopy Sciences) for 10 min and washed for 5 min with distilled water. Nuclei were counterstained for 1 min with a haematoxylin solution, according to Mayer (3870, J. T. Baker, Center Valley, PA, USA). Then, the samples were washed, dehydrated with xylene, and coverslipped with the Eukitt mounting medium (03989, Merck Millipore).
Schiff s reagent
Manufactured by Electron Microscopy Sciences
Schiff's reagent is a chemical solution used in histological and cytological staining techniques. It is a colorless liquid that turns pink or purple in the presence of aldehydes, which are commonly found in various biological samples. The primary function of Schiff's reagent is to serve as a staining agent, allowing the visualization and identification of specific cellular structures or components that contain aldehydes.
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Lab products found in correlation
Eukitt mounting medium, by Merck Group (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Pro series 3ccd camera, by Olympus (1 mentions)
Eukitt mounting medium, by Electron Microscopy Sciences (1 mentions)
Alcian blue, by Electron Microscopy Sciences (1 mentions)
Hemodi, by Thermo Fisher Scientific (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Harris hematoxylin, by Merck Group (1 mentions)
Ds fi1, by Nikon (1 mentions)
5 protocols using schiff s reagent
1
PAS Staining of Hippocampal Sections
2
Quantification of Rat Goblet Cells
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Four sections from each rat were stained as previously described (Trevizan et al., 2016 ). Briefly, sections were subjected to a series of deparaffinization, stained with Alcian Blue (Electron Microscopy Sciences) for 30 min, washed with tap and distilled water, treated with 0·5% periodic acid (Electron Microscopy Sciences), washed with distilled water for 2 min, stained with Schiff’s Reagent (Electron Microscopy Sciences) for 20 min, washed with tap water for 5 min, stained with haematoxylin (1 min), washed with tap water for 2 min, dehydrated, cleared in HemoDi (Fisher Scientific, Pittsburgh, PA) and mounted in Eukitt Mounting Medium (Electron Microscopy Sciences). Eight images from each section were taken with a digital camera (Pro series 3CCD camera) coupled to an optical microscope (Olympus BX50, Microscope Central, Feasterville, PA) with a 20x objective. The number of goblet cells presented in 0·96 mm2 in the mucosa of each animal were quantified using Image Pro Plus software (Media Cybernetics, Rockville, MD).
3
Quantification of Apoptotic Pericytes in Retinal Tissue
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To analyze ACs and PL, RTD preparations representing the three groups of rats were stained with PAS and Harris hematoxylin (Sigma-Aldrich, St. Louis, MO). The RTD slides were immersed in 0.5% PAS (Sigma-Aldrich) for 10 min, rinsed in dH2O, and exposed to Schiff's reagent (Electron Microscopy Sciences, Hatfield, PA) as previously described [10 (link)]. Next, the slides were rinsed in dH2O and immersed in Harris hematoxylin (Sigma-Aldrich) for 20 s. After rinsing in dH2O, the slides were subjected to dehydration through an ethanol gradient and clearing in xylene and then were mounted in mounting medium (Permount; Fisher Scientific, Pittsburgh, PA). Ten representative 36 μm × 27 μm images were photographed using a digital camera (DS-Fi1; Nikon, Tokyo, Japan) connected to a computer, and the images were analyzed for ACs and PL. Apoptotic pericytes were identified as PL (pericyte ghosts) based on prominent histological characteristics, including basement membrane protrusion as an empty shell. Capillaries devoid of pericytes and endothelial cells were considered ACs. Counts were also scored by a second independent examiner in a masked manner. The data represent the average of both scores.
4
Periodic Acid-Schiff Staining Protocol
5
Alcian Blue-PAS Staining of Colon Tissue
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Alcian blue-PAS staining was performed on paraffin sections of colon tissue fixed with Carnoy's fixative. Before performing Alcian blue-PAS assay, 5 μm colon tissue sections were baked overnight at 55°C. Colon tissue sections were deparaffinized in xylene, dehydrated with 100% ethanol. The air-dry slides were stained with Alcian blue solution (1 g of Alcian blue, pH 2.5, 3 mL/L of acetic acid, and 97 mL of distilled water) for 30 min for goblet cells. This was followed by rinsing in tap water for 10 min, oxidizing in periodic acid (5 g/L) for 5 min, rinsing in tap water for 10 min, and staining in Schiff's reagent as a counter stain (Electron Microscopy Sciences, Cat. NO. 26853-01, Hatfield, PA, USA) for 10 min. After washing in distilled water for 10 minutes, dehydrated through ethanol and xylene, and cover‐slipped using a permount (Fisher Scientific, Cat.No. SP15-100, Waltham, MA, USA).
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