Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

Immunofluorescence staining of KRT5 in sections of formalin-fixed paraffin-embedded human skin (positive) and human cerebellum (negative)*. Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with sodium citrate (pH6.0) for 20 minutes. The section was then incubated at room temperature for 1 hour with ab321868 at 1/500 dilution and then incubated for 1 hour with ab150133 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green). 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. This data was developed using the same antibody clone in a different buffer formulation. *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

Immunofluorescence staining of TSG101 in sections of formalin-fixed paraffin-embedded human colon*. Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with sodium citrate (pH6.0) for 20 minutes. The section was then incubated at room temperature for 1 hour with ab320807 at 1/100 dilution and then incubated for 1 hour with ab150133 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green). 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. This data was developed using the same antibody clone in a different buffer formulation. *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• Flow Cyt

Lab

Flow Cytometry - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

Flow cytometric analysis of 4% Paraformaldehyde fixed, 90% Methanol permeabilized MCF7 (Human breast adenocarcinoma epithelial cell) / THP-1 (Human monocytic leukemia monocyte) cells labelling Iba1 with ab289874 at 1/1000 compared with a Goat IgG /Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Donkey anti-Goat IgG (Alexa Fluor^®488, ab150133) at 1/2000 was used as the secondary antibody.

Negative control :  MCF7 (PMID : 30386176).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

ab321868 staining KRT5 in A431 (positive) and MCF7 (negative) cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal donkey serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab321868 at 1µg/ml (shown in green) and ab202272, Alexa Fluor® 594 Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (shown in pseudocolour magenta). Cells were then incubated with Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

This data was developed using the same antibody clone in a different buffer formulation.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

ab323241 staining SQSTM1 in wild-type Hap1 cells and SQSTM1 knockout Hap1 cells +/- Chloroquine (50uM 24 hours) cells. The cells were fixed with 100% methanol (5 min) permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal donkey serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4° C with ab323241 at 5ug/ml (shown in green) and ab202272 Alexa Fluor® 594 Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (shown in pseudocolour magenta). Cells were then incubated with ab150133 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

ab320059 staining ACTA2 in HeLa (positive) and A-431 (negative) cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal donkey serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab320059 at 1µg/ml (shown in green) and ab202272, Alexa Fluor® 594 Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (shown in pseudocolour magenta). Cells were then incubated with ab150133 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) treated with 500 ng/ml Trichostatin A for 4 hours (Red) / Untreated control (Green) cells labelling alpha Tubulin (acetyl K40) with ab289875 at 1/1000 compared with a Goat IgG / Black isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody).

Donkey anti-Goat IgG (Alexa Fluor® 488, ab150133) at 1/2000 was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

ab320807 staining TSG101 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal donkey serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab320807 at 1µg/ml (shown in green) and ab202272, Alexa Fluor® 594 Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (shown in pseudocolour magenta). Cells were then incubated with Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

ab320807 staining TSG101 in NIH/3T3 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal donkey serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab320807 at 1µg/ml (shown in green) and ab202272, Alexa Fluor® 594 Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (shown in pseudocolour magenta). Cells were then incubated with ab150133 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue). Also suitable in cells fixed with 4% paraformaldehyde (10 min). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling non-muscle Myosin IIB/MYH10 with ab300649 at 1/1000 dilution (0.1ug) (Red) compared with a Goat IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Donkey anti-Goat IgG (Alexa Fluor® 488, ab150133) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Rat primary neural/glia cell cells labelling GFAP with ab302644 at 1/1000 dilution (0.1ug)/ Right (Red) compared with a Goat IgG / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Donkey anti-Goat IgG (Alexa Fluor® 488, ab150133) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Mouse primary neural/glia cell cells labelling GFAP with ab302644 at 1/1000 dilution (0.1ug)/ Right (Red) compared with a Goat IgG / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Donkey anti-Goat IgG (Alexa Fluor® 488, ab150133) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

ab323241 staining SQSTM1 in NIH/3T3 +/- chloroquine (50uM 24 hours) cells. The cells were fixed with 100% methanol (5 min) permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal donkey serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4° C with ab323241 at 1ug/ml (shown in green) and ab202272 Alexa Fluor® 594 Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (shown in pseudocolour magenta). Cells were then incubated with ab150133 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

ab320059 staining ACTA2 in SV40LT-SMC cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal donkey serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab320059 at 1µg/ml (shown in green) and ab202272, Alexa Fluor® 594 Rabbit monoclonal [EP1332Y] to alpha Tubulin - Microtubule Marker (shown in pseudocolour magenta). Cells were then incubated with ab150133 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

Immunofluorescence staining of KRT5 in sections of formalin-fixed paraffin-embedded human skin (positive) and human cerebellum (negative)*. Performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with sodium citrate (pH6.0) for 20 minutes. The section was then incubated at room temperature for 1 hour with ab321868 at 1/500 dilution and then incubated for 1 hour with ab150133 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green). 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. This data was developed using the same antibody clone in a different buffer formulation. *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)

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. ab150133 Alexa Fluor® 488 donkey anti-goat IgG (H+L) (green) 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.

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

• Alexa Fluor®

Unknown

Alexa Fluor® - Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150133)