The procedure was performed as previously described [24 (link)]. Retina was fixed in 2% PFA and 2.5% glutaraldehyde (Merck KGaA, Darmstadt, Germany), and prepared for semi-thin (0.5 μm) section on Lecia microtome (Germany). Then the semi-thin sections were stained with 1% toluidine blue (Bio-Rad, Life Science Research, Hercules, CA, US) for 1 minute, followed by section dehydration and mounting before visualization under light microscope (Zeiss, Germany).
Toluidine blue
Manufactured by Bio-Rad
Sourced in United States
Toluidine blue is a metachromatic dye used in various laboratory applications. It is primarily utilized for staining and visualization purposes, highlighting specific cellular or tissue structures. The core function of toluidine blue is to provide a clear and contrasting staining of samples, enabling better observation and analysis under a microscope.
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Lab products found in correlation
2 protocols using toluidine blue
1
Retina Tissue Preparation for Microscopy
2
Polyphosphate Granules Visualization in C. albicans
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The presence of intracellular polyP granules was determined by light microscopy by Neisser staining of C. albicans cells (Gurr, 1965 ). Paraformaldehyde-fixed cells (Enjalbert et al., 2006 (link)) were mounted onto a slide and stained with freshly prepared solution A (methylene blue, 0.1% [Sigma-Aldrich]; glacial acetic acid, 5%; ethanol, 5%) and solution B (crystal violet, 10% in ethanol) for 10–15 s. Slides were rinsed with water, and solution C (chrysoidin Y, 1% [Sigma-Aldrich]) was added for 45 s and rinsed off, and slides were allowed to dry. DIC images were captured using a Zeiss Axioscope with a 63× oil immersion objective and AxioVision imaging system.
For urea-PAGE and toluidine blue staining, RNA was extracted as described previously (Smith et al., 2004 (link)), as this procedure also releases polyP. Total RNA (20 μg) containing polyP was resolved by electrophoresis on 15% polyacrylamide TBE-urea gels (Bio-Rad, Hercules, CA) in 1× Tris-borate-EDTA (TBE) buffer. Gels were then fixed with methanol and glycerol, stained in toluidine blue (Sigma-Aldrich), and destained as described previously (Smith and Morrissey, 2007 (link)).
For urea-PAGE and toluidine blue staining, RNA was extracted as described previously (Smith et al., 2004 (link)), as this procedure also releases polyP. Total RNA (20 μg) containing polyP was resolved by electrophoresis on 15% polyacrylamide TBE-urea gels (Bio-Rad, Hercules, CA) in 1× Tris-borate-EDTA (TBE) buffer. Gels were then fixed with methanol and glycerol, stained in toluidine blue (Sigma-Aldrich), and destained as described previously (Smith and Morrissey, 2007 (link)).
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