Donkey Anti-Goat IgG H&L (Alexa Fluor® 405) preadsorbed (ab175665)

ICC/IF image of ab7291 stained HeLa cells. The cells were 100% methanol fixed (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then incubated in 1%BSA / 0.3M glycine in 0.1% PBS-Tween for 1h to block non-specific protein-protein interactions. The cells were then incubated with the primary antibody (ab7291, 1µg/ml) overnight at +4°C. Ab98800, goat anti-mouse IgG, was then added as a secondary bridging antibody, at 1/250 dilution for 1h. Ab175665 Alexa Fluor® 405 Donkey polyclonal Secondary Antibody to Goat IgG - H&L pre-adsorbed (blue) was then used at 1µg/ml for 1h as a tertiary antibody. DRAQ5™ (ab108410) was used to stain the cell nuclei (red) at a concentration of 1.25µM.

The negative control (inset) is a secondary-only assay to demonstrate low non-specific binding of the secondary antibody.

Postnatal day 6 mouse testes were fixed with 4% paraformaldehyde. Tissue was embedded in O.C.T. and frozen. 5 micron sections were cut and transferred to slides. Sections were permeabilized with 0.1% Triton X-100 in PBS, and blocked with 3% BSA in 0.1% Triton X-100 + PBS. Sections were incubated with either (A) no primary antibody or (B) Goat Anti-DDX4 at a 1/800 dilution for 1 h at RT. Sections were then washed 3X with 0.1% Triton X-100 in PBS and Donkey Anti-Goat IgG H&L (Alexa Fluor^® 405) (ab175665) applied at a 1/500 dilution. Sections were then mounted after washing 3X with 0.1% Triton X-100 in PBS.