Oil Red O stain
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This protocol is for lipid and fat staining on frozen sections. It may not suitable for paraffin embedded tissue sections.
Fixation: 10% formalin (fixation is necessary, damage of unfixed sections has been seen)
Section: Frozen sections at 5-10 um thick
1) Cut fresh frozen tissue sections at 5-10 um thick and mount on slides.
2) Air dry slides for 30-60 minutes at room temperature and then fix in ice cold 10% formalin for 5-10 minutes. Air dry again for another 30-60 minutes or rinse immediately in 3 changes of distilled water.
3) Let slides air dry for a few minutes.
4) Place in absolute propylene glycol for 2-5 minutes to avoid carrying water into Oil Red O.
5) Stain in pre-warmed Oil Red O solution for 8-10 minutes in 60 ºC oven.
6) Differentiate in 85% propylene glycol solution for 2-5 minutes.
7) Rinse in 2 changes of distilled water.
8) Stain in Gill's or Mayer's hamatoxylin for 30 seconds.
9) Wash thoroughly in running tap water for 3 minutes.
10) Place slides in distilled water.
11) Mount with glycerin jelly or other aqueous mounting medium.
Nuclei: pale blue