Cy3 conjugated donkey anti mouse igg secondary antibody

For the IF examination, 1 × 10⁵ cultured cells on coverslips were fixed with methanol for 10 min at −20 °C and incubated with primary antibodies against CCR5 (1:25, #sc-32304; Santa Cruz Biotechnology), CCL3 (1:100, #LS-C384561; LifeSpan BioSciences), and CD204 (1:100, #SRA-E5; TransGenic, Kobe, Japan) at 4 °C overnight. The cells were then incubated with AlexaFluor-488® conjugated donkey anti-rabbit secondary antibody, AlexaFluor-488® conjugated donkey anti-mouse secondary antibody, and Cy3-conjugated donkey anti-mouse IgG secondary antibody (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA). The nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI, 1:1000; Vector Laboratories, Burlingame, CA). Images were taken with a Zeiss LSM 700 laser-scanning microscope and analyzed using the LSM software ZEN 2009 (Carl Zeiss, Oberkochen, Germany).

To evaluate the relationship between Calb, Parv, and SMI-32, cerebellar sections were mounted onto positive charged slides, pretreated with boiling citric acid (pH6) for 10 min for antigen retrieval, washed with TBS, blocked in a TBS/0.5%Triton solution containing 3% donkey serum, and dual-labeled with rabbit anti-Calb and mouse anti-Parv or mouse anti-SMI-32. Sections were incubated overnight for Calb at RT washed with TBS/1% donkey serum and incubated in Cy2-conjugated donkey anti-rabbit immunoglobulin G (IgG) secondary antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. After several washes with TBS/1% donkey serum, sections were incubated overnight using either anti-Parv or anti-SMI-32 at RT and then placed in Cy3-conjugated donkey anti-mouse IgG secondary antibody for 1 h (1:200, Jackson ImmunoResearch). Following development, sections were washed in TBS, dehydrated in graded alcohols, cleared in xylenes, and cover-slipped with DPX mounting medium. Fluorescence was visualized with the aid of a Revolve Fluorescent Microscope (Echo Laboratories, San Diego, CA, USA) with excitation filters at wavelengths 489 and 555 nm for Cy2 and Cy3, respectively.

To assess differentiation of C2C12 cells and primary myoblasts, immunohistochemistry was performed using anti-myosin heavy chain antibody (MF-20) from DHSB, which recognizes the fast and slow sarcomeric myosin heavy chains. The antibody was incubated overnight, followed by a Cy3-conjugated donkey anti-mouse IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. DAPI (0.5 μg/ml) counterstain was used to label chromatin. Pictures were taken of a minimum of six random field of view at × 10 magnification per well. The differentiation index is defined as the number of myosin heavy chain positive nuclei divided by the total number of nuclei. The fusion index is described as the number of myosin heavy chain positive nuclei in myotubes divided by the total number of myotubes.