Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120)

ab6326 staining BrdU in Hela cells. Untreated and BrdU treated (10uM for 24 hours) cells. The cells were fixed with 100% methanol (5 min) and then subjected to acid hydrolysis using 2M HCL in 0.1% PBS-Tween for 30 minutes at room temperature to denature the DNA. They were then permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1hr. The cells were then incubated overnight at 4°C with ab6326 at 1μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150165, Goat polyclonal Secondary Antibody to Rat IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

ICC/IF image of ab7291 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7291, 1µg/ml) overnight at +4°C. The secondary antibody (orange) was ab150120 Alexa Fluor® 594 goat anti-mouse IgG (H+L) used at 1µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Cross-reactivity of the polyclonal secondary antibody ab182017 was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards at 1 µg/ml (50µl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. ab182017 was then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Donkey anti-Goat IgG H&L (HRP) (ab6885) was used at 1/10,000 dilution (50 µl/well), followed by incubation for 1h at RT.

Fot the batch tested, ab182017 showed a cross-reactivity below 2% towards Chicken IgY, 6% towards Human IgG, 7% towards Rabbit IgG and 47% towards Rat IgG.

This data was developed using the unconjugated antibody (ab182017).

Cross-reactivity of Goat anti-Mouse IgG H&L (ab182017) and Goat anti-Mouse IgG H&L obtained from two different vendors was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards (Rabbit, Human, Mouse and Rat) at 1 µg/ml (50µl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. Secondary antibodies were then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Donkey anti-Goat IgG H&L (HRP) (ab6885) was used at 1/10,000 dilution (50 µl/well), followed by incubation for 1h at RT. This data is from a representative dilution.

This data was developed using the unconjugated antibody (ab182017).

Image: Courtesy of Dr. Shaohua Li, UMDNJ-Robert Wood Johnson Medical School

Sample: mouse embryonic stem cell-differentiated embryoid bodies (EBs)

Preparation:

Fix in 3%PFA in PBS for 30 min at RTIncubate in 7.5% sucrose-PBS for 3h at RTIncubate in 15% sucrose-PBS at 4 degree Celsius overnightEmbed the EBs in tissue-Tek OCT compoundCut frozen sections to 4-20 µm thickness

Primary antibody 1: Rabbit anti-laminin, 1:400

Primary antibody 2: Mouse anti-disabled-2, 1:100
Secondary antibody 1: Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488) pre-adsorbed (ab150081), 1:200

Secondary antibody 2: Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) pre-adsorbed (ab150120), 1:200
Nuclei were counterstained with DAPI

Unpurified ab134175 staining Cyclin D1 in MCF7 (Human breast adenocarcinoma cell line) cells treated with KN-93 (ab120980).
The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab134175 at 10µg/ml and ab7291 at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an Goat anti-Rabbit Alexa 488 secondary (ab150081) at 2 µg/ml (shown in green) and Goat anti-Mouse Alexa 594 secondary (ab150120) at 2 µg/ml (shown in pseudo color red). Nuclear DNA was labeled in blue with DAPI.
Negative controls: 1, Rabbit primary and anti-mouse secondary antibody; 2, Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.