Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

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 / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab7291, 10μg/ml) overnight at +4°C. The secondary antibody (red) was ab150119 Alexa Fluor^® 647 goat anti-mouse IgG (H+L) used at 2μg/ml for 1h. 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.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Immunofluorescence staining of Collagen alpha-1(I) chain 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 ab317799 at 1/500 dilution, and then incubated for 1 hour with ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 dilution (shown in red). 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® 647) preadsorbed (AB150119)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells labelling T2A + P2A with ab269488 at 1/100 (9.29 ug/ml) dilution, followed by ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (ab150119) antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic and nuclear staining in HEK-293T cells transfected with a P2A expression vector containing a GFP tag is observed. The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (ab150119) at 1/1000 2 ug/ml dilution.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells labelling T2A + P2A with ab269488 at 1/100 (9.29 ug/ml) dilution, followed by ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (ab150119) antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic and nuclear staining in HEK-293T cells transfected with a T2A expression vector containing a GFP tag is observed. The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (ab150119) at 1/1000 2 ug/ml dilution.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab115730 staining SLC2A1 in wild-type A549 cells, with negative expression in SLC2A1 knockout A549 cells. The cells were fixed with 100% methanol (5 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab115730 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in 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.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab92513 staining ERG in THP-1 cells, with negative expression in HCT116 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab92513 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in 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.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab92547 staining VIM in wild-type HeLa cells with negative expression in VIM knockout HeLa cells. The cells were fixed with 4% formaldehyde (10 min) permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab92547 at 2 μg/ml and ab7291 Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081 Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488) pre-adsorbed at 1/1000 dilution (shown in green) and ab150119 Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 647) pre-adsorbed at 1/1000 dilution (shown in 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.This product also work with 100% methanol (5 min) fixation under the same testing conditions.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab216576 staining DUSP4 in wild-type A549 cells, with negative expression in DUSP4 knockout A549 cells. The cells were fixed with 100% methanol (5 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab216576 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in 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

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab125011 staining TSG101 in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab125011 at 1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in red). 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.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab222754 staining SERPINE1 in HUV-EC cells, with negative expression in HEK293 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab222754 at 0.1 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in 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.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab52625 staining KRT19 in MCF7 cells, with negative expression in SHSY5Y cells. The cells were fixed with 100% methanol (5 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab52625 at 2 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in 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. This product also work with 4% formaldehyde (10 min) fixation under the same testing conditions.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized p53 KO HAP1 (human p53 knockout chronic myelogenous leukemia near-haploid cell, Left) / Parental HAP1 (Right) cells labelling p53 with ab308609 at 1/1000 dilution (0.1 ug) (Red) compared with a Mouse monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Mouse IgG (Alexa Fluor® 647, ab150119) at 1/5000 dilution was used as the secondary antibody.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Composite multiplex immunofluorescence staining of SYP, MAP2 and Tau (MBD region) 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 ab309493 at 1µg/ml dilution (shown in green), ab318993 at 1µg/ml (shown in magenta), and ab308439 at 1µg/ml (shown in yellow). Then incubated for 1 hour with ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed 1/1000, ab150086 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) preadsorbed 1/1000, and ab150173 Goat Anti-Chicken IgY H&L (Alexa Fluor® 488) 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® 647) preadsorbed (AB150119)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK293-GFP-LRRK2 cells labelling RAB10 (phospho T73) with ab315172 at 1/400 dilution, followed by ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (ab150119) antibody at 1/1000 2 ug/ml dilution (Green).

Increased number of strongly stained cytoplasmic foci after Doxycycline treatment compared to untreated cells and after MLi-2 treatment as expected. Images were acquired on the Perkin Elmer® Operetta HCA and a maximum intensity projection of 8 confocal planes is shown for the representative images.

ab202272 Recombinant Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab202272) was used to counterstain tubulin at 1/200 2.5 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (ab150119) at 1/1000 2 ug/ml dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK293-GFP-LRRK2 cells labelling RAB10 (phospho T73) with ab315174 at 1/400 dilution, followed by ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (ab150119) antibody at 1/1000 2 ug/ml dilution (Green).

Increased number of strongly stained cytoplasmic foci after Doxycycline treatment compared to untreated cells and after MLi-2 treatment as expected. Images were acquired on the Perkin Elmer® Operetta HCA and a maximum intensity projection of 8 confocal planes is shown for the representative images.

ab202272 Recombinant Alexa Fluor® 594 Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab202272) was used to counterstain tubulin at 1/200 2.5 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (ab150119) at 1/1000 2 ug/ml dilution.

• Flow Cyt

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Flow Cytometry - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Flow Cytometry analysis of HCT 116 (Human colorectal carcinoma epithelial cell, Left) / MCF7 ( Human breast adenocarcinoma epithelial cell, Right) cells stained for MUC1 using ab70475 at a 1/1000 dilution (0.1 μg) (Red) compared to Mouse monoclonal IgG - Isotype Control (Black) and cells without incubation with primary antibody and secondary antibody (Blue). Negative control; HCT 116. (PMID : 14998492)

• Flow Cyt

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Flow Cytometry - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Flow Cytometry analysis of human peripheral blood mononuclear cell (PBMC) treated with 100ng/ml GM-CSF and 50ng/ml IL-4 for 3 days then change new medium (100ng/ml GM-CSF and 50ng/ml IL-4) for another 3 days (right panel) compared to mouse monoclonal IgG isotype control (left panel). ab123494 used at a 1/400 dilution. Secondary is Goat Anti-Mouse IgG (Alexa Fluor® 647, ab150119) used at a 1/2000 dilution. Gated on viable cells. Bright secondary antibody like Alexa Fluor®647 conjugated ones are recommended to get desirable signal.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab208992 staining mutant p53 in A431 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab208992 at 0.2µg/ml (pseudocoloured in green) and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 dilution (shown in red). 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.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab20181 staining HLA DR in Raji cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised in 0.1% Triton X-100 for 5 minutes 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 overnight at +4°C with ab20181 at 1/1000 dilution and ab7291, Mouse monoclonal to alpha Tubulin at 1/1000 dilution. Cells were then incubated with ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed, at 1/1000 dilution (shown in red) and ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488), preadsorbed at 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab208992 staining wildtype p53 in Hek293 cells (a high expressing cell line). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab208992 at 0.2µg/ml (pseudocoloured in green) and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 dilution (shown in red). 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.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

ab208992 staining mutant p53 in wild-type Hap1 cells (top panel) and p53 knockout Hap1 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab208992 at 0.2µg/ml (pseudocoloured in green) and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 dilution (shown in red). 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.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

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 EDTA (Ph9.0) using retrieval settings of 100°C for 40 minutes. The section was then incubated at room temperature for 1 hour with ab323239 at 5µg/ml dilution (shown in green), ab300645 at 5µg/ml (shown in magenta), and ab178846 at 5µg/ml (shown in yellow). Then incubated for 1 hour with ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed 1/1000, ab150084 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed 1/1000, and ab150173 Goat Anti-Chicken IgY H&L (Alexa Fluor® 488) 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.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Immunofluorescence staining of Histone H3 (acetyl K27) in sections of formalin-fixed paraffin-embedded human colon (positive). 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 ab317802 at 1/100 dilution, and then incubated for 1 hour with ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 dilution (shown in red). 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.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Immunofluorescence staining of Histone H3 (tri methyl K9) 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 ab317800 at 1/500 dilution, and then incubated for 1 hour with ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 dilution (shown in red). 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® 647) preadsorbed (AB150119)

ab223844 staining MLH1 in wild-type Hap1 cells, with negative expression in MLH1 knockout Hap1 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.1% Triton x-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab223844 at 0.2 μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 0.5 μg/ml. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150119, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 647), pre-adsorbed at 1/1000 dilution (shown in 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

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Immunofluorescence staining of SOX9 using ab185230 in primary hippocampal mouse neurons/glia (obtained from Transnetyx Tissue by BrainBits LLC cat.no. C57EHP) DIV14. The cells were fixed with 4% formaldehyde (10 min) permeabilized with 0.1% TritonX-100 (in PBS) for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab185230 at 5 μg/ml, ab4674 (anti-GFAP) at 1/1000 dilution and ab104224 (anti-NeuN) at 1/1000 dilution. Cells were then incubated with ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) ab150176 Goat Anti-Chicken IgY H&L (Alexa Fluor^® 594) preadsorbed (shown in red) and ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 647) (shown in purple) all secondary antibodies at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).
As expected most GFAP positive cells are also SOX9 positive while NeuN positive cells are SOX9 negative.
Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Immunofluorescence staining of Histone H3 (acetyl K27) in sections of formalin-fixed paraffin-embedded rat kidney (positive). 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 ab317802 at 1/100 dilution, and then incubated for 1 hour with ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 dilution (shown in magenta). 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Immunofluorescence staining of SOX9 using ab196450 in primary hippocampal mouse neurons/glia (obtained from Transnetyx Tissue by BrainBits LLC cat.no. C57EHP) DIV14. The cells were fixed with 4% formaldehyde (10 min) permeabilized with 0.1% TritonX-100 (in PBS) for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab196450 at 1 μg/ml (shown in green), ab4674 (anti-GFAP) at 1/1000 dilution and ab104224 (anti-NeuN) at 1/1000 dilution. Cells were then incubated with ab150176 Goat Anti-Chicken IgY H&L (Alexa Fluor^® 594) preadsorbed (shown in red) and ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 647) (shown in purple) all secondary antibodies at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).
As expected most GFAP positive cells are also SOX9 positive while NeuN positive cells are SOX9 negative.
Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Immunofluorescence staining of SOX9 using ab185966 in primary hippocampal mouse neurons/glia, (obtained from Transnetyx Tissue by BrainBits, LLC, cat.no. C57EHP), DIV14. The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% TritonX-100 (in PBS) for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab185966 at 1 μg/ml, ab4674 (anti-GFAP) at 1/1000 dilution and ab104224 (anti-NeuN) at 1/1000 dilution. Cells were then incubated with ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green), ab150176, Goat Anti-Chicken IgY H&L (Alexa Fluor® 594) preadsorbed (shown in red) and ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (shown in purple), all secondary antibodies at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue). As expected, most GFAP positive cells are also SOX9 positive, while NeuN positive cells are SOX9 negative. SOX9 positive cells, which are not GFAP positive (e.g. asterisk) are likely neural stem cells/ oligodendrocyte precursor cells present in the culture. Images were acquired with the Perkin Elmer Operetta HCA and a maximum intensity projection of confocal sections is shown.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

Immunofluorescence staining of Tyrosine Hydroxylase in sections of formalin-fixed paraffin-embedded rat brain (positive) and rat liver (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 ab317796 at 1/500 dilution, and then incubated for 1 hour with ab150119 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 dilution (shown in red). 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.

• sELISA

Lab

Sandwich ELISA - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)

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® 647) preadsorbed (AB150119)

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®

Unknown

Alexa Fluor® - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed (AB150119)