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

Immunofluorescence. CCR2, CCR5, CXCR2, and CXCR4 were detected in brain sagittal slices of 6-monthold SD and taiep rats ( = 3 per each group). Rats were deeply anesthetized with chloral hydrate and perfused through the ascending aorta with 100 mL of PBS 1x and then by 150 mL of 4% paraformaldehyde in PBS. Their brains were removed and maintained in the fixative for 48 h at 4 ∘ C. Each brain was included in paraffin and was cut into 3 m slices on the sagittal plane using a Leica RM 2135 microtome (Leica Microsystems, Nussloch, Germany). Slices were individually collected on a glass slide. Tissue slices (previously deparaffinized) were rehydrated and incubated with 0.5% IgG-free bovine serum albumin in PBS-Tween 20 (0.1%) for 20 min at RT. Slices were incubated with rabbit monoclonal antibodies to CCR2, CCR5, CXCR2, or CXCR4 (1 : 500 dilution, Abcam Inc., Cambridge, MA, USA) at 4 ∘ C overnight. The secondary antibody was a goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC). The counterstaining was made using DAPI or propidium iodide (2 g/mL). Tissue slices were mounted on glass slides using VECTASHIELD (Vector Laboratories, Burlington, Ontario, Canada). The fluorescence within the cells was analyzed with 5x and 40x objectives of a Leica DMIRE2 microscope using the filters A for DAPI, K3 for FITC, and TX2 for propidium iodide (Leica Microsystems, Wetzlar, Germany). The images were digitalized with a Leica DC300F camera (Leica Microsystems, Nußloch, Germany) and processed with a workstation Leica FW4000, version V1.2.1 (Leica Microsystems Vertrieb GmbH, Bensheim, Germany). Samples incubated with only the secondary antibodies were used as negative controls.