Examining Ubiquitinated Protein Aggregates in Tissue Sections - Protein Misfolding and Cellular Stress in Disease and Aging - page 169

2. Materials

2.1. Deparaffinization/
Rehydration of
Sections

1. Xylenes certified A.C.S X5-1 (Fisher Chemicals).
2. Ethanol, anhydrous denatured, histological grade (100%,
diluted with deionized water to 95% and 70%).
3. Deionized water (dH 2 O).
4. 7 glass slide staining dishes, with covers and one glass slide
rack (Fisher Scientific); 2 dishes contain xylenes bath, 2 dishes
contains 100% ethanol bath, one dish contains 95% ethanol
bath, one dish contains 70% ethanol bath, and one dish con-
tains dH 2 O bath (See Note 1).
5. Permeabilization buffer: Phosphate-buffered saline (PBS ++ )
(1× DPBS/Modified + Calcium + Magnesium, HyClone Thermo
Scientific) with 0.1% Triton X-100.

2.2. Antigen Retrieval

1. Sodium citrate buffer: 10 mM Tri-sodium citrate dihydrate,
0.05% Tween 20, pH 6.0. To make 1 L, weigh 2.94 g of
sodium citrate and mix with distilled water to dissolve. Adjust
pH to 6.0 with 1 N HCl and then add 0.5 ml Tween 20 and
mix well. Add distilled water to 1 L. Store at room tempera-
ture for 3 months.
2. Pyrex microwave-safe glass baking dish (7 × 5 × 1.5 in.) with a
large glass plate used as a cover.
3. Forceps.
4. Plastic 5-place slide mailer containers, end opening (Fisher).
5. Washing buffer: PBS ++ .

1. Blocking solution: 10% normal goat serum (Wisent) and 0.2%
saponin (Calbiochem) in PBS ++ . Make fresh as required. 10%
saponin stock can be stored at −20°C and added to a final of
0.2%.
2. Primary antibody: Mouse monoclonal antibody FK2 (Biomol)
(See Note 2).
3. Secondary antibody: Goat anti-mouse AlexaFluor 568
(Molecular Probes).
4. Nuclear stain: 300 nM DAPI (4,6-diamidino-2-phenylindole)
in water.
5. Mounting media: Dako Fluorescent Mounting Media
(Dako).
6. Microscope cover glass (circular, 25 mm, 0.13-0.17 mm
thick) (Fisher).
7. Pap pen (Invitrogen).

2.3. Staining