Examining Ubiquitinated Protein Aggregates in Tissue Sections - Protein Misfolding and Cellular Stress in Disease and Aging - page 173

4. Notes
1. Xylenes, ethanol, and ethanol dilution baths should be
replaced after approximately 5 to 7 uses. Use a fresh dH 2 O
bath with every experiment.
2. The FK2 primary antibody recognizes mono- and poly-
ubiquitinated proteins but not free ubiquitin. It is therefore
ideal for detection of ubiquitinated-protein aggregates in
the cell.
3. A deparaffinization step is not needed if frozen tissue sections
are available. Once applied to a glass microscope slide, the
frozen tissue sections can be simply incubated in the permea-
bilization buffer and the rest of the protocol can be carried
out.
4. In some tissues, in order to unmask the ubiquitin antigen
within aggregates, heat-induced epitope retrieval is done by
boiling samples in sodium citrate buffer using a microwave.
Use a sufficient volume of sodium citrate buffer to cover the
slides by a least a few cm to allow for evaporation during boil-
ing. The dish should be large enough to allow the slides to sit
at the bottom.
5. Do not leave the microwave on continuously because the tis-
sues may bubble off the slides.
6. In our experience, some tissues (i.e., pancreas, gut) do not
require antigen unmasking in order to visualize ubiquitinated
aggregates. In these cases, incubation in blocking solution
will suffice. The blocking solution contains saponin, a mild
detergent that further permeabilizes the tissue so that anti-
gens can be revealed.
7. The amount of primary antibody added is just enough to
cover the tissue sample, between 100 and 500 ml, depending
on how big your tissue section is on the slide. The pap pen
barrier around the sample will retain this volume. Since the
dilution buffer contains saponin, a surfactant, the added pri-
mary antibody should spread over and cover the tissue sec-
tion. Another antibody of different species lineage can also
be added at this time, to compare localization of two
proteins.
8. Adding blocking solution establishes a good meniscus on top
of the tissue section.
9. Do not expose the secondary antibody to light for extended
periods of time. Another secondary antibody specific to
the primary can also be added at this time, to compare
localization.