Biology Reference
In-Depth Information
residual food and/or chyme may be cleaned off using a cotton tip applicator
dipped in PBS.
2.
With the lumen facing upward, use a clean, glass microscope slide to gently
scrape the mucosal layer of the intestine. There will be a thin layer of intestine
left on the glass plate. The intestinal mucosa will mostly stick on the glass slide.
The mucosal scrapings will include enterocytes, as well as other epithelial cell
types and the submucosa.
3.
Aliquot intestinal mucosa scrapings into 1.5-ml microcentrifuge tubes (or 2-ml
cryotubes) and freeze rapidly in liquid nitrogen. Intestinal mucosa scrapings
from a 5 cm section can be aliquoted into two or three tubes with ample tissue
for TAG, mRNA, and/or protein analysis. Store samples at
80
C until
analysis.
4.
Add 1 ml of 1 M Tris-HCl pH 7.4 to an intestinal mucosa sample collected in
step 3.
5.
Homogenize the intestine sample in the same tube as collected with a polytron
homogenizer until smooth, approximately for 15 s. Remove 300
m
lofthe
homogenate and place in a 15 ml glass test tube for lipid extraction. Remove
10
m
l of the homogenate and place in a 1.5 ml microcentrifuge tube for protein
analysis.
6.
Add 5 ml HIP and 1 ml of milli-Q water to the 15 ml glass test tube containing
the intestine homogenate. Vortex for 30 s every 10 min, for 30 min. Allow the
mixture to separate at room temperature.
7.
Remove the upper layer (organic phase) and place in a clean, 15-ml test tube.
8.
Re-extract the intestinal homogenate by adding 4 ml HIP to the remaining
aqueous layer and vortex for 30 s. Allow the mixture to separate at room
temperature.
9.
Remove the upper layer (organic phase) and combine with the previously
extracted upper layer for that sample.
10.
Dry the upper layer under nitrogen flow in a 50
C water bath. A white or
clear film will develop on the bottom of the glass test tube. This is the extracted
lipid.
11.
Redissolve the lipid in 400
l of isopropanol. Vortex lightly to remove any lipid
film attached to the side of the tube.
12.
Use 10
m
l (in duplicate) for determination of TAG concentration using a Wako
L-Type TGMKit. Use the “Microtiter Procedure” provided byWako and dilute
standards in isopropanol. Keep in mind that samples collected from mice fed a
high fat diet or gavaged with oil may need to be further diluted to be in range for
the TAG kit.
13.
Use 10
m
m
l of the intestinal mucosa homogenate (saved at step 5) for
determination of protein concentration by the BCA protein assay. This sample
will likely need to be diluted 10-fold in order to be in range for the BCA kit.
9.1.3.1.3
Results
Using the results of TAG and protein concentrations, calculate the mg of TAG per
mg of protein.
