Dear Ridvan, have you prepared the new stock for ORO? You need to crush the powder very well. You can gently heat after mixing it and filter it later. You can apply this solution for staining.

• I didn't prepare new stock. I used Oil Red O Stain kit of Atom Scientific brand. For this protocol:

1. Rinse slides in distilled water 2. Rinse with 60% isopropyl alcohol to clear background 3. Stain with freshly prepared Oil Red O working solution 15 mins 4. Rinse briefly with 60% isopropyl alcohol. Carefully rinse in distilled water. 5. Lightly stain nuclei with alum haematoxylin for 10 seconds 6. Rinse with distilled water and blue 7. Mount in aqueous mountant.

I stained mammarian gland with the same kit also and I saw that dye molecules were scattered. I added an image concerning with this tissue. Thank you.

Thank you. I Will try this protocol.

Hi do more "rinses" with the 60% isopropanol and (or) extend the time of the "rinse" . You can also do a delipidised negative control, use acetone to remove the lipid from a section. That way you can check for background.. It may just be background precipitate of the ORO solution when you change to the next reagent which is water any remaining staining solution will drop its ORO dye at that point...

I tried protocol Mohammed Mahdy was suggested and lipit droplets was stabilized in their own places. I added an image of fatty liver tissue. Thank you very much.

Thank you very much mohamed Mahdy.

Hi Mohamed,

I think your protocol would work great for my tissues. However, before I move forward, do you know how or if your protocol would affect other staining outcomes using the frozen tissue? If not do you know someone I may contact to ask further questions?

Thank you!

Hi Mohamed Mahdy,

I need to change my question because I already froze my tissues so it is too late for me to do the protocol you did. With my frozen (-80) livers I did the following to stain with Oil-Red-O:

1) Cryostat liver slides cut at 6 microns

2) Fix slide in formalin for 30 seconds

3) Oil-Red-O for 20 minutes

4) Rinse

5) Haematoxylin for 20 seconds

6) Rinse

7) Immu-mount the slides

Is there an other way to preserve the lipid droplets after they have already been frozen in a -80 freezer? I was going to test Peter Walker's protocol to see how that goes but since that is very similar to what Ridvan Bağci did initially I am thinking I may have similar results to what I do now which is lipid droplets broken out of the cells.

Thank you Mohamed Mahdy. Your protocol can be something for me to try in the future.