Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) preadsorbed

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) preadsorbed (AB150118)

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 (yellow) was ab150118 Alexa Fluor^® 555 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) preadsorbed (AB150118)

Composite multiplex immunofluorescence staining of APP, PSD95 and Tau staining in a section of formalin-fixed paraffin-embedded human Alzheimer’s brain*.

Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with EDTA (Ph9.0) using retrieval settings of 100°C for 40 minutes. The section was then incubated at room temperature for 1 hour with ab201060 at 1µg/ml dilution (shown in green), ab317793 at 1µg/ml (shown in magenta), and ab317841 at 1µg/ml (shown in yellow). Then incubated for 1 hour with ab150118 Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) preadsorbed 1/1000, ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 1/1000, and ab150135 Donkey Anti-Goat IgG H&L (Alexa Fluor® 647) preadsorbed 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Dako Fluorescence Mounting Medium ^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

For other IHC staining systems (automated and non-automated), customers should optimize variable parameters such as antigen retrieval conditions, antibody concentrations and incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) preadsorbed (AB150118)

HeLa cells showing negative staining by ICC/IF using only secondary antibody. 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 secondary antibody (yellow) was ab150118 Alexa Fluor^® 555 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.

• sELISA

Lab

Sandwich ELISA - Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) preadsorbed (AB150118)

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.

For 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 were developed using the unconjugated antibody (ab182017).

• sELISA

Lab

Sandwich ELISA - Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) preadsorbed (AB150118)

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.

These data were developed using the unconjugated antibody (ab182017).

• Alexa Fluor®

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Alexa Fluor® - Goat Anti-Mouse IgG H&L (Alexa Fluor® 555) preadsorbed (AB150118)