Examining Ubiquitinated Protein Aggregates in Tissue Sections - Protein Misfolding and Cellular Stress in Disease and Aging - page 170

3. Methods

Under diabetic stress, ubiquitinated-protein aggregates can be
seen within a variety of different tissues ( 3 ). Depending on the
tissue, antigen-retrieval steps may have to be performed in order
to visualize the aggregates. The following method is specific for
paraffin-embedded tissue. During the antigen-retrieval step,
methylene bridges formed by formalin are broken and this in turn
exposes antigenic sites.

3.1. Preparing Tissue
Sections for
Immunofluorescence

1. These instructions assume the samples are fixed paraffin-
embedded tissue, which is mounted onto 25 × 75-mm glass
microscope slides. A ventilated fume hood should be used
when working with xylenes. Place slides in the glass rack and
submerge the rack into the first glass dish containing xylenes.
Incubate for 10 min. Once done, submerge the slides into
the second glass dish containing xylenes for another 10 min.
A third wash is done by submerging the slides into the first
dish containing xylenes for a final 10 min.
2. Incubate slides in two washes of 100% ethanol for 5 min each.
3. Incubate slides in one wash of 95% ethanol for 5 min.
4. Incubate slides in one wash of 70% ethanol for 5 min.
5. Incubate slides in one wash of dH 2 O for 5 min.
6. Incubate slides in permeabilization buffer within plastic slide
mailer containers for 15 min. (See Note 3)
7. Pour off permeabilization buffer and wash sections twice with
PBS ++ for 2 min each, gently inverting with each wash.

3.2. Antigen Retrieval

1. Add the prepared sodium citrate buffer to the glass micro-
wave-safe Pyrex dish covering approximately 3 cm from the
bottom of the dish (See Note 4). Place the dish in the micro-
wave and add a glass plate to cover the dish. Ideally, the
microwave should be used inside a ventilated fume hood.
Microwave the buffer until boiling. Once boiled, carefully
remove the glass cover and very gently, using forceps, ease the
slides (tissue side up) into the dish, allowing them to settle at
the bottom.
2. Place the dish back in the microwave, add the cover and start a
timer for 30 min. Begin to microwave the samples at a low
power for a couple of minutes. Keep watching the samples, if
you notice boiling in the dish, immediately stop the microwave
and let the samples sit for 1 min (See Note 5). Then start the
microwave again at a low setting for another 2 min, allowing
the samples to sit for 1 min intervals. Two minutes is only sug-
gested as a starting point. A control experiment is recommended