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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Donkey Anti-Goat IgG H&L (Alexa Fluor® 555) preadsorbed (AB150134)

Composite multiplex immunofluorescence staining of Iba1, GFAP and MAP2 staining in a section of formalin-fixed paraffin-embedded human cerebral cortex*.

Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with Sodium citrate (Ph6.0) using retrieval settings of 100°C for 20 minutes. The section was then incubated at room temperature for 1 hour with ab300156 at 1µg/ml dilution (shown in green), ab183830 at 1µg/ml (shown in magenta), and ab302644 at 1µg/ml (shown in yellow). Then incubated for 1 hour with ab150161 Goat F(ab')2 Anti-Rat IgG Fc (Alexa Fluor® 488) preadsorbed 1/1000, ab150083 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 647) preadsorbed 1/1000, and ab150134 Donkey Anti-Goat IgG H&L (Alexa Fluor® 555) 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 the EVOS™ S1000 Spatial Imaging System (ThermoFisher Scientific) with spectral unmixing and minor subsequent contrast adjustment.

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 - Donkey Anti-Goat IgG H&L (Alexa Fluor® 555) preadsorbed (AB150134)

HeLa cells showing negative staining by ICC/IF using only tertiary antibody. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. ab150134 Alexa Fluor® 555 donkey anti-goat IgG (H+L) (yellow) was then used at 2µg/ml for 1h as a tertiary antibody. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Donkey Anti-Goat IgG H&L (Alexa Fluor® 555) preadsorbed (AB150134)

ICC/IF image of ab7291 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 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. ab98800, goat anti-mouse IgG, was then added as a secondary bridging antibody, at 1/250 dilution for 1h. ab150134 Alexa Fluor® 555 donkey anti-goat IgG (H+L) (yellow) was then used at 2µg/ml for 1h as a tertiary antibody. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• Alexa Fluor®

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Alexa Fluor® - Donkey Anti-Goat IgG H&L (Alexa Fluor® 555) preadsorbed (AB150134)