Examining Ubiquitinated Protein Aggregates in Tissue Sections - Protein Misfolding and Cellular Stress in Disease and Aging - page 172

11. DAPI is added for 10 min at room temperature to identify
the nuclei.
12. Wash section in dH 2 O 5 times at 10 min each and then
aspirate dry from one corner.
13. Using Dako mounting media, drop a small amount onto a
circular glass coverslip and apply directly onto the exposed
tissue. Apply a small amount of pressure to the coverslip and
then allow the slide to dry at room temperature in a dark
drawer. The sample can be viewed under the microscope once
the mounting media has dried. The sample can also be stored
in the dark at −20°C for months.
14. The slides can be observed using confocal microscopy.
Excitation at 578 nm induces the AlexaFluor 568 conjugate
fluorescence exhibiting ubiquitinated aggregates as red struc-
tures in the tissue, whereas excitation at 364 nm induces
DAPI fluorescence and the nucleus is seen in blue. OpenLab
software can be used to analyze tissue and overlay fluorescent
images. Ubiquitinated-protein aggregates occurring in diabetic
pancreatic tissue can be seen by the arrows in Fig. 2 ( 3 ).
Fig. 2. Zucker diabetic fatty rat pancreatic tissue sections exhibit ubiquitinated protein
aggregates in vivo. Representative images of pancreatic sections isolated from 19-week-
old Zucker diabetic fatty rats (ZDF) and 6-week-old ZDF rats. Sections were stained
using mAb FK2 (identifies mono-and polyubiquitin proteins) to examine ubiquitinated
protein aggregates, and an antibody against insulin to identify b -cells within islets and
a nuclear DAPI stain. Ubiquitinated-protein aggregates were observed in pancreatic
b -cells of the 19-week-old ZDF rat as shown by the arrows. These aggregates were not
evident in the control 6-week-old ZDF tissue. All pancreatic tissues contained a small
number of infiltrating immune cells that stain nonspecifically with secondary antibodies
as shown by the arrowhead. Scale bar, 10 m m.