*4.6. Immunocytochemistry*

Cells were seeded over glass coverslips (10 mm diameter) in 6-well plates at a density of 50,000 cells/well in a final volume of 2 mL of medium. Once the treatments were concluded, cells were washed three times with PBS and fixed in bouin solution for 15 min. After fixation, cells were washed three times and then permeabilized by incubation with 1% Triton X-100 at room temperature for 15 min. Nonspecific binding was blocked by incubation with bovine serum 30 min at room temperature. Incubation with anti-human Apo D antibody 1:2000 (provided by Dr. Carlos López-Otín, department of Biochemistry

and Molecular Biology, University of Oviedo; see [58,77,78]) was carried out overnight in a humid chamber at 4 ◦C. After three washes in PBS, coverslips were incubated 30 min at room temperature using a biotinylated horse universal antibody (Universal quick, PK-8800, Vector Laboratories, Inc., Burlingame, CA, USA) diluted 1:50. After that, cells were incubated with streptavidin Alexa Fluor® 550 conjugate (1:500; S2138, Invitrogen, Paisley, Scotland, UK). Finally, cells were washed in distilled water, dehydrated, cleared in eucalyptol and mounted with Fluoromount. The fluorescence was visualized in a Nikon Eclipse E400 microscope equipped with a Nikon G2-A and recorded by a digital camera (Nikon DN-100). The resulting immunocytochemical signal was selected with Photoshop and quantified with ImageJ 1.57 software (NIH, Bethesda, MD, USA) [79]. Images were acquired under the same conditions of illumination, diaphragm and condenser adjustments, exposure time, and background correction. For control purposes, representative cell cultures were processed in the same way with a nonimmune serum or with specifically absorbed sera instead of the primary antibody. Under these conditions no specific immunostaining was observed.
