RE: [Histonet] Oil Red Staining

From:"Jacqui Detmar"

Hi Jennifer.  As luck would have it, I just did an Oil Red stain on my
mouse placentae the other day.  The protocol I use was taken from IHC
World.  I'm often trying to determine whether a placental phenotype
exists in different mouse knockout strains, and this is one that I apply
to see how lipid metabolism is going.  Anyway, here it is:

Fix tissue in 10% formaldehyde (I don't like to use the ready-made 10%
formalin b/c I'm afraid the added methanol will affect the lipid levels.
So, I make fresh solution every time, using 37% formaldehyde and 0.1 M
phosphate buffer.

Wash in PBS.  Cryoembed in OCT.

Cut 8-10 micron sections.

Allow sections to dry at room temperature (about 15-30 minutes)

Rinse off OCT in ddH2O (about 2 changes)

Place in 100% propylene glycol for 5 minutes.

Stain in Oil Red O for 8 minutes at 60 degrees Celsius

Rinse in 85% propylene glycol for 5 minutes.

Rinse in ddH2O for 3-5 minutes.

Do *light* stain with hematoxylin (I use Harris, but the original
protocols called for Mayer's or Gill's; Also, I apply the hematoxylin
with a dropper to the section, count to 5 (Mississippi) and squirt the
stain off with water in a squirt bottle).

Blue in 1% NaHCO3

Wash in running water for 2-3 minutes.

Mount with glycerin jelly or other aqueous mountant.  Actually, I use a
70% glycerol solution, drop the coverslip on, allow the slide to dry
overnight and apply clear nail-polish around the edges the next day.

Some of this might sound a bit amateurish for those who do full-scale
histology, so please feel free to make suggestions/comments about this
protocol.  The focus of my work is not actually histology, but I like to
implement histological techniques in my characterization of knockout
tissue.  I find it is very informative and oddly enough, highly
under-used by my colleagues who also study targeted gene knockout mice!
How can *anybody* resist pretty pictures!!??? .

Anyway, here is the recipe for the (0.5%) Oil Red O stain:

0.5 g Oil Red O
100 mL propylene glycol

Add small amount of propylene glycol to Oil Red O, with stirring.
Gradually add remaining propylene glycol.  Heat until solution reaches
95 degrees Celsius.  Filter through coarse paper while warm.  Let stand
overnight at room temperature.  Filter again.  Solution can be kept at
RT for years and years and year.

If you need any more info, please feel free to contact me.


Jacqui Detmar, Post-doctoral Fellow
Samuel Lunenfeld Research Institute, room 876
Mount Sinai Hospital
600 University Avenue
Toronto, ON, Canada
M5G 1X5

phone:   416-586-4800 x2451/x2290
fax:        416-586-8588

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