Analysis of Chemokines and Receptors Expression Profile in the Myelin Mutant Taiep Rat

Soto-Rodriguez, Guadalupe; Gonzalez-Barrios, Juan-Antonio; Martinez-Fong, Daniel; Blanco-Alvarez, Victor-Manuel; Eguibar, Jose R.; Ugarte, Araceli; Martinez-Perez, Francisco; Brambila, Eduardo; Millán-Perez Peña, Lourdes; Pazos-Salazar, Nidia-Gary; Torres-Soto, Maricela; Garcia-Robles, Guadalupe; Tomas-Sanchez, Constantino; Leon-Chavez, Bertha Alicia

ELISA was used to determine the protein levels of CCL2, CCR1, CCR2, CCL5, CCR5, CCR8, CXCL1, CXCR2, CXCR4, FGF2, and VEGFA in homogenates obtained from the brainstem of SD or taiep rats at 1 and 6 months of age ( = 5 per age in each group), as described previously [4] . The protein content was determined using the method by Sedmak and Grossberg [25] . Aliquots containing 5 g of total protein were placed into wells of ELISA plates to determine chemokines and receptors in separate assay. Then, proteins were denatured by the addition of 100 L of 0.1 M carbonate buffer (pH 8.0) added into each well and the plate was incubated for 18 h at 4 ∘ C. To block nonspecific binding sites, 200 L of 0.5% bovine serum albumin, IgG-free, was added to each well at room temperature (RT). After 30 min of incubation, the wells were washed thrice with PBS-Tween 20 (0.1%). Rabbit monoclonal antibodies to CCL2, CCR1, CCR2, CCL5, CCR5, CCR8, CXCL1, CXCR2, CXCR4, FGF2, and VEGFA (1 : 500 dilution, Abcam Inc., Cambridge, MA, USA) detect both native and denatured proteins. They were added into each well and incubated for 2 h at RT. After three washings with PBS, a horseradish peroxidase-conjugated goat anti-rabbit IgG (1 : 1000 dilution, Dako North America Inc., Carpinteria, CA, USA) was added and incubated for 2 h at RT. The antigen-antibody complex was revealed by adding 100 L of 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) containing 0.3% H 2 O 2 into each well. After 15 min, the optical density (OD) was determined at 415 nm using a Benchmark multiplate reader (Bio-Rad, Hercules, CA, USA) as was described elsewhere [4] .