Biology Reference
In-Depth Information
5.
Liquid nitrogen or dry ice
6.
Superfrost Plus microscope slides
7.
Cover slips and mounting medium
8.
Cryostat
9.
Oil Red O stock solution (stable for 6 months)
Oil Red O: 0.5 g
Isopropanol: 100 ml
10.
Oil Red O working solution (make fresh)
Stock Oil Red O solution: 24 ml
Distilled water: 16 ml
Combine the stock solution and the distilled water. Let the mixture stand for
10-15 min, then filter using a 0.2
m
m syringe filter to remove undissolved
particulates before use.
11.
60% isopropanol in water (wash solution)
9.1.2.2.2
Methods
1.
For the best results, cardiac perfusion fixation of the mouse should be done
using 10% formalin. However, the desired portions of small intestine can be
excised and incubated in 10% formalin for 2 h at room temperature.
2.
After fixing, the lumen of the small intestine is cleaned and tissue stored in 10%
sucrose in PBS at 4
C overnight.
3.
Cut the small intestine tissue into 5-7 mm pieces and embed with Tissue-Tek O.
C.T. compound using desired mold and orientation. Snap-freeze molds
containing tissue and O.C.T. and store at
80
C until use.
4.
Using a cryostat, prepare 10-12
m frozen sections and mount on Superfrost
Plus microscope slides. Sectioned tissues can be stored at
m
20
C.
5.
Remove histological sections on slides from the freezer. Allow to dry and return
to room temperature. Rehydrate sections in distilled water for 5 min. Blot excess
distilled water.
6.
Dip slides in 60% isopropanol for 30 s using 5-place slide mailers or 50 ml
conical tubes.
7.
Dip slides into Oil Red O working solution for 15 min.
8.
Destain tissue with 60% isopropanol for 30 s.
9.
Rinse under running tap water for 2 min.
10.
Counterstain with hematoxylin, if desired.
11.
Cover slip using desired mounting medium.
12.
Image using a light microscope.
9.1.2.3
Results
Figure 9.3
shows Oil Red O images of the jejunum (section 2) from two different
high fat-fed male mice. The left panel is from a mouse that has euthanized in the
fasted state. The right panel is from a high fat-fed mouse that has been euthanized
in the fed state (2 h after the start of the light cycle). In these images, the CLDs are
stained red.
