[Histonet] validating oil red o staining

[Geoff McAuliffe]

Hi Jeremy:

    Interesting question. First, my experience with lipid staining is 
minimal so intrepret my suggestions accordingly.

Jeremy Browne wrote:

> Clarification,
>
> Thanks for all your responses.  Its clear that I haven't explained 
> myself very well at all:
>
> We would like to use oil red O to quantify lipid localisation in 
> frozen sections of hepatic and muscle tissue.  However, we must first 
> demonstrate that the amount of staining we get (density) is in fact 
> equal to the amount of lipid present in the sections and to do this we 
> need to set up some titrations using positive controls with known and 
> different concentrations of lipid to show a linear response between 
> staining intensity and lipid concentration.
>
    Is it known that Oil Red O has a linear reaction with the lipids in 
question (do you know the identity of the lipids in question)? What does 
the literature say on this topic? Have you tried a model system in which 
varing amounts of  the lipids in question are spotted on paper or gels 
and the linearity of Oil Red O staining is investigated?

>  However, a fat smear is difficult to use as a positive control in 
> this way because it simply washes off the slides.  Therefore, we would 
> like to know what others have done to demonstrate linearity with the 
> oil red O staining procedure.  Is there a way we can treat the fat to 
> overcome its liquid nature (that is to say not the lipid in the muscle 
> and liver but the actual 'fat' being used as a positive control)?
>
    How about cutting serial sections and staining one with Oil Red O 
and using some biochemical method to quantify the lipids you 
extract/wash off from the adjacent section? Spectrophotmetery? Of 
course, the method you use to extract the lipids would have to be 
specific to the Oil Red O stained lipids or those lipids would have to 
be a very arge majority of the lipids present.
    This project may keep you busy for awhile.

Geoff

-- 
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff <@t> umdnj.edu
**********************************************