As part of my master's degree, I am working on a research project to cultivate fat lobules from explant tissue. We would like to study cell viability and functionality of fat cells over a period of 14 days in vitro and are looking for easily feasible light microscopy methods that are not as labor intensive as the preparation of sections.
I am looking forward to your answers!
Michael Ruegenberg
Osmium tetroxide paraffin procedure for fat. After fixation of a small piece about 0.0.5 × 0.5 × 0.5 cm sample in 10% Neutral buffer formalin, they placed in osmium tetroxide for demonstration of fat by the method that allows paraffin-embedded in the tissue
1. Chaco2589 from: https://www.quizlet.com/13184659/ht-08-connective-and-muscle-tissue-flash-cards/ (2012). 2. Gates, L., Adler, R. R. & Elangbam C. S. Osmium tetroxide post-fixation and periodic acid-Schiff dual-staining technique to demonstrate intracellular lipid and glycogen in the mouse liver section – a novel method for co-visualization of intracellular contents in paraffin-embedded tissue, J Histotechnol, 39(1), 2–7 (2016). https://doi.org/10.1080/01478885.2015.1106072. 3. Abd-Elhafeez, H. H. et al. Migratory activities and stemness properties of rodlet cells. Microsc. Microanal. https://doi.org/10.1017/
S1431927620001828
4-Lisa G, Rick RA & Chandikumar SE (2016). Osmium tetroxide post-fixation and periodicacid-Schiff dual-staining technique to demonstrate intracellular lipid andglycogen in the mouse liver section – a novel method for co-visualization ofintracellular contents in paraffin-embedded tissue. J Histotechnol 39(1), 2–7
Methods:
Osmium Tetroxide Paraffin Procedure for Fat
I. The purpose of the stain: Demonstration of fat by a method that allows paraffin embedded of the tissue.
II. The principle of the stain: Osmium tetroxide chemically binds with the fat blackening it in the process. This is the only method for demonstration of fat on paraffin sections.
III. Fixatives: 10% NBF.
IV. Sectioning: Stain the wet tissue with the Osmium tetroxide, then process the tissue. The section must be no thicker than 2 mm or the osmium will not penetrate.
V. Controls: Most tissue contains fats, normally no control is needed.
VI. Reagents:
1. 1% Osmium tetroxide solution: osmium tetroxide & distilled water.
2. 0.5% Periodic acid solution: periodic acid & distilled water.
VII. Procedure:
1. Trim formalin fixed tissue to 2 mm thickness, then wash thoroughly for at least 30 minutes.
2. Rinse the tissue well in distilled water.
3. Place the section in a small quantity of Osmium Tetroxide solution and leave for at least 1 to 2 hours.
4. Rinse slides in two changes of distilled water for 15 minutes each.
5. Differentiate slides by placing them in the 0.5% Periodic acid solution for 30 minutes.
6. Wash slides in tap water for 30 minutes.
7. Process routinely, beginning with 70% alcohol, embed as usual.
8. Cut paraffin sections 4-5 microns, pick on slides and dry as normal.
9. Deparaffinize and hydrate as usual.
10. Counterstain with hematoxylin, Masson Trichrome.
11. Dehydrate, clear and mount slides as normal.
IX. Results:
Fat .............................................................. Black
Other tissue elements ................................. According to method used
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