We follow the instruction from Biovision to stain lipid droplets in endometrial cancer cells, however, we didn't get any staining. We need help to trouble shooting it. Here is our protocol.
ORO Staining
Note: All components: For 12-well plate, use 1ml per well.
1. Cell Fixing: Remove media from cells and gently wash 2X with PBS.
2. Add Formalin (10%) to each well and incubate for 30 min.
a. Note: Do not pipet directly onto cells, pipet to the side of well or plate and mix by rotating.
3. Cell Staining: Prepare Oil Red O Working Solution.
4. Remove formalin and gently wash cells 2X with dH2O.
5. Make Isopropanol (60%) by adding 3 parts Isopropanol (100%) to 2 parts of water. Add Isopropanol (60%) to each well and incubate for 5 min.
6. Remove isopropanol and add Oil Red O Working Solution to completely and evenly cover the cells. Rotate plate or dish and incubate for 10 min.
7. Remove Oil Red O solution and wash 2-5X with dH2O as needed until excess stain is no longer apparent. Add Hematoxylin and incubate for 1 min.
8. Remove Hematoxylin and wash with dH2O 2-5X as needed. Keep cells covered with dH2O at all times and while viewing under microscope. Lipid droplets appear red and nuclei appear blue.
a. Note: Discard used Oil Red O Solution. Re-use of Oil Red O Solution results in poor staining quality.
Thank you.
how did you make your Formalin 10% is one step please check. Formaldehyde is around 40%.
if you make t3n % of this actually one might be using 4% formaline. IN a control one must get colour to know if the protocol is staining the lipid droplets.
As beifre adding the stains only thing you are adding in 10% formalin i thought of highlighting this.
i agree, use 4% formaldehyde. Make this fresh from paraformaldehyde powder in PBS. Rinse well in PBS to remove all fixative before staining.
Hi Michael and Ellen, thank for your kind response and your suggestions. Now, I know the fixation is very important. I got 10% new balanced formalin from our Comparative Pathology lab. We didn't make it freshly. I am confusing with the answer, should we use formalin or formaldehyde? And what percentage? We usually use 4% fromaldehyde to fix cells for immunostaining. Thank you again to help us out.
Please use 4% formaldehyde. Without good fixation it's difficult to get results. Ellen already pointed out that good washing ought to be there.
Dilute formaldehyde is called formalin.
If possible use normal healthy tissue for controls, even fresh animal tissues available in market say chicken, fish etc.
(I only teach Histopathology in theory, as trained person not available. My understanding was conceptual only. ) It's really nice Dr Ellen added her comment.
Michael Durairaj S , thank you. Do you by chance know why 10% formaline don't do job for fixation? Is it because it is not made freshly? Or concentration is not ideal. Thank you again to help us out.
Madam i posted a message in the inbox. sorry about the mistyping in the first message.
10% Formalin should do the job.
Fixation time the number of hours.
Temperature at which fixation is taking place lower temperature
normally a local lab processing tissue for fixation one can ask the concerned. in general all printed protocols work.
Some odd reason if the formalin is less than 10% (4% formaldehyde) it will take longer duration to fix. Same is if temperatures are lower it will take longer time.
initially stating something new few hitches will be there. My understanding is even a old formalin should give good results. Always good to make ones own solutions for experiments.
expect for impurity ideally fixation should take place.
Michael Durairaj S , we used 10% formalin, but we didn't get any ORO labeling. We are willing to try 4% formaldehyde.
Here it says fixing time can be increased from 30 min to 1 hour.
More importantly the ORO stain should be freshly prepared . 15 minutes before. IS STABLE FOR TWO HOURS.
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