For routine histology, tissue was either fixed in 4% paraformaldehyde (30 min) and embedded in paraffin or embedded and frozen in optimal cutting temperature (OCT) medium. Five µm thick sections were cut and stained as described previously [24 (link)]. Briefly, sections were stained with hematoxylin (Haematoxylin Gill III, Surgipath, Leica, Germany) for 2 min and 1% eosin Y (Surgipath, Leica, Germany) for 1 min to observe the gross tissue architecture and degree of decellularization. To visualize the proteoglycan content, cryosections were stained with 1% alcian blue (Morphisto, Offenbach am Main, Germany) for 30 min together with a counterstain of nuclear fast red (Morhphisto, Offenbach am Main, Germany). For glycoproteins, PAS staining was performed using 1% periodic acid (Honeywell Fluka, Charlotte, NC, USA) for 10 min, and Schiff reagent (Roth, Karlsruhe, Germany) for 90 s. Samples were examined using either a Hamamatsu NanoZoomer S60 (Hamamatsu Photonics, Herrsching, Germany) or a bright field fluorescence microscope (Axio Imager.A1, Zeiss) and images were processed using ProgRes CapturePro Software (JENOPTIK, Dreseden, Germany)).
Schiff reagent
Manufactured by Carl Roth
Sourced in United States
Schiff reagent is a chemical solution used in analytical chemistry. It is primarily used to detect the presence of aldehydes in chemical samples.
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2 protocols using schiff reagent
1
Histological Evaluation of Decellularized Tissues
2
Periodic Acid-Schiff Staining of Paraffin Sections
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Tissue samples were fixed and embedded in paraffin. For analysis, 4-mm-thick slides were treated in xylol for paraffin removal, then rehydrated using successive treatment with 100%, 95%, 70%, and 50% ethanol solution and H 2 O for 3 minutes each.
For periodic acid-Schiff staining, slides were incubated for 5 minutes with 0.8% periodic acid to achieve the oxidation reaction. Afterward the slides were rinsed with distilled water three times and incubated for 15 minutes with Schiff reagent (Carl Roth) at room temperature. The slides were washed with tap water two times for 5 minutes. Counterstaining was performed by using Mayer's Hematoxylin Solution (Sigma-Aldrich, Munich, Germany). After the final washing step with tap water, the slides were covered with glycerol/gelatin (Sigma-Aldrich, Munich, Germany).
For periodic acid-Schiff staining, slides were incubated for 5 minutes with 0.8% periodic acid to achieve the oxidation reaction. Afterward the slides were rinsed with distilled water three times and incubated for 15 minutes with Schiff reagent (Carl Roth) at room temperature. The slides were washed with tap water two times for 5 minutes. Counterstaining was performed by using Mayer's Hematoxylin Solution (Sigma-Aldrich, Munich, Germany). After the final washing step with tap water, the slides were covered with glycerol/gelatin (Sigma-Aldrich, Munich, Germany).
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