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

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

ab96462 staining Nup153 in NIH3T3 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 goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab96462 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150084, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution (shown in pseudocolour 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-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

ab14734 staining VDAC1 in HEK293 (positive) and HEK293-VDAC1 KO (negative) cells. The cells were fixed with 100% methanol (5 min), 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 1h. The cells were then incubated for 2 hours at room temperature with ab14734 at 1µg/ml (shown in green) and ab186735, anti-TOMM20 antibody [EPR15581-54] - Mitochondrial Marker (shown in magenta) and ab323238, anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker – Chicken IgY Chimeric (shown in yellow) . Cells were then incubated with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution; ab150084, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution; and ab150175, Goat Anti-Chicken IgY H&L (Alexa Fluor® 647) 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.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-937 (human histiocytic lymphoma monocyte) cells labeling SIGLEC5 with ab307846 at 1/50 dilution (9.72 µg/ml) followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 µg/ml) (Green). Confocal image showing membranous and cytoplasmic staining in U-937 cell line. Negative control : K-562 (PMID : 9731071). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/ml) (Red). The nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 µg/ml).

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

Immunofluorescence staining of E-Cadherin using ab231303 in wild-type A431 cells (top panel) and CDH1 knockout A431 cells (bottom panel). The cells were fixed with 4% formaldehyde (10 min), permeabilized 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 with ab231303 at 1.0 µg/mL and ab6046 at 1 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (ab150084) (shown in red), both at 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a confocal section is shown.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

ab57632 staining VPS35 in wild-type HAP1 cells (top panel) and VPS35 knockout HAP1 cells (bottom panel). The cells were fixed with 4% PFA (10 min), permeabilized 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 with ab57632 at 0.2 µg/mL and ab6046 at 1 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor® 488) (ab150117) (shown in pseudo colour green) and goat secondary antibody to Rabbit IgG (Alexa Fluor® 594) (ab150084) (shown in pseudo colour red) both at 1/1000. Nuclear DNA was labelled with DAPI (shown in pseudo colour blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

Immunofluorescence staining of E-Cadherin using ab231303 in wild-type A431 cells (top panel) and CDH1 knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized 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 with ab231303 at 1.0 µg/mL and ab6046 at 1 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (ab150084) (shown in red), both at 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a confocal section is shown.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

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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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

Immunofluorescence staining of Ctip2 using ab18465 in Jurkat cells (+ve expression control top panel) and Daudi cells (-ve expression control bottom panel). The cells were fixed with 4% formaldehyde (10 min) permeabilized 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 with ab18465 at 0.2 µg/mL and ab6046 at 1 µg/mL overnight at +4°C followed by a further incubation at room temperature for 1h with Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150165) (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150084) (shown in red) both at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

Immunofluorescence staining of Ctip2 using ab18465 in Jurkat cells (+ve expression control top panel) and Daudi cells (-ve expression control bottom panel). The cells were fixed with 100% methanol (5 min) permeabilized 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 with ab18465 at 0.2 µg/mL and ab6046 at 1 µg/mL overnight at +4°C followed by a further incubation at room temperature for 1h with Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150165) (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150084) (shown in red) both at 1/1000 dilution. Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Rat liver (fresh) tissue labeling Pan Cav2 with ab315092 at 1/50 (10.32 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/mL) dilution (Green).

Negative control : confocal image showing no staining on rat liver. The section was incubated with ab315092 for 60 mins at room temperature. The section was then mounted using Fluoromount®.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbedat 1/1000 (2 ug/mL) dilution.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

ab96462 staining Nup153 in Rin-5F 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 goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab96462 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150084, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution (shown in pseudocolour 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

Composite multiplex immunofluorescence staining of ab279297, ab317042 and ab308439 staining NeuN, NEFH and Tau (MBD region) in Mouse Primary Neurons DIV14 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 ab279297 (shown in green), ab317042 (shown in magenta) and ab308439 (shown in yellow) at 1µg/ml. Cells were then incubated with ab150161 Goat F(ab')2 Anti-Rat IgG Fc (Alexa Fluor® 488) preadsorbed, ab150175 Goat Anti-Chicken IgY H&L (Alexa Fluor® 647) preadsorbed and ab150084 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) 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.

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

ICC/IF image of ab6046 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 (ab6046, 1μg/ml) overnight at +4°C. The secondary antibody (orange) was ab150084 Alexa Fluor® 594 goat anti-rabbit 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-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)

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 : Mouse anti-Ki67 (ab53280), 1 : 50

Primary antibody 2 : Rabbit anti-laminin, 1 : 400
Secondary antibody 1 : Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 488) pre-adsorbed (ab150117), 1 : 200

Secondary antibody 2 : Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) pre-adsorbed (ab150084), 1 : 300
Nuclei were counterstained with DAPI

This image is courtesy of Dr. Shaohua Li

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Alexa Fluor® - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (AB150084)