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

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

Immunofluorescence staining of SPP1 in sections of formalin-fixed paraffin-embedded human kidney (positive) and human testis (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 ab322993 at 1/500 dilution and then incubated for 1 hour with ab150165 Goat Anti-Rat 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.

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

ab322910 staining Ctip2 in Jurkat (positive) and Daudi (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 goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab322910 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 ab150165, Goat Anti-Rat 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 confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

Immunofluorescence staining of FOS in sections of formalin-fixed paraffin-embedded human breast cancer tissue (positive) and human normal breast tissue (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 ab322911 at 1/100 dilution and then incubated for 1 hour with ab150165 Goat Anti-Rat 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. *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

ab322993 staining SPP1 in Hep G2 +/- Brefeldin (300ng/ml, 3 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 goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab322993 at 5µg/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 ab150165, Goat Anti-Rat 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

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

Lab

Immunocytochemistry/ Immunofluorescence - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

ab6326 staining BrdU in Hela cells. Untreated and BrdU treated (10uM for 24 hours) cells. The cells were fixed with 100% methanol (5 min) and then subjected to acid hydrolysis using 2M HCL in 0.1% PBS-Tween for 30 minutes at room temperature to denature the DNA. They were then 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 1hr. The cells were then incubated overnight at 4°C with ab6326 at 1μg/ml and ab7291 Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150165 Goat polyclonal Secondary Antibody to Rat IgG - H&L (Alexa Fluor^® 488) pre-adsorbed at 1/1000 dilution (shown in green) and ab150120 Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594) pre-adsorbed at 1/1000 dilution (shown in pseudocolour 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

Immunofluorescence staining of Ctip2 in sections of formalin-fixed paraffin-embedded human tonsil (positive) and human skeletal muscle (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 ab322910 at 1/100 dilution and then incubated for 1 hour with ab150165 Goat Anti-Rat 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. *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

Dot plot showing BrdU-treated HeLa cells stained with ab6326. Cells were incubated with 10 μM BrdU for 30 minutes prior to being harvested washed twice in 1x PBS and fixed in 70% ethanol (4°C added drop-wise) for at least 30 minutes on ice. Once fixed pellets were acid denatured with 2M HCl for 30 minutes at 22°C and then neutralised with borate buffer (0.1M pH8.5).

Samples were washed and incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab6326 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used wasGoat Anti-Rat IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150165) at 1/2000 dilution for 30 min at 22°C.

7-AAD was added to cells 20 min prior to data acquisition.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) with 530/30 and 685/35 bandpass filters.

• Flow Cyt

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Flow Cytometry - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

Overlay histogram showing HeLa cells stained with ab19136 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab19136, 1μg/1x10^6 cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rat IgG (H&L) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rat IgG2a [aRTK2758] (ab18450, 2μg/1x10^6 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

ab90247 staining F4/80 in Raw264.7 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 overnight at 4°C with ab90247 at 5µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150165, Goat polyclonal Secondary Antibody to Rat IgG - H&L (Alexa Fluor® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

Immunofluorescence staining of Ctip2 in sections of formalin-fixed paraffin-embedded mouse brain (positive) and mouse skeletal muscle (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 ab322910 at 1/100 dilution and then incubated for 1 hour with ab150165 Goat Anti-Rat 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

Immunofluorescence staining of FOS in sections of formalin-fixed paraffin-embedded mouse brain (positive) and mouse 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 ab322911 at 1/100 dilution and then incubated for 1 hour with ab150165 Goat Anti-Rat 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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

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 (green) was ab150165 Alexa Fluor® 488 goat anti-rat IgG (H&L) used at 2µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• IHC-Fr

AbReview

Immunohistochemistry (Frozen sections) - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

Postnatal day 6 mouse testes were fixed with 4% paraformaldehyde. Tissue was embedded in O.C.T. and frozen. 5 micron sections were cut and transferred to slides. Sections were permeabilized with 0.1% Triton X-100 in PBS, and blocked with 3% BSA in 0.1% Triton X-100 + PBS. Sections were incubated with either (A) no primary antibody or (B ) anti-KIT (ab65525) for 1 h at RT. Sections were then washed 3X with 0.1% Triton X-100 in PBS and Goat-Anti Rat 488 (ab150165) applied at a 1/500 dilution. Sections were then mounted after washing 3X with 0.1% Triton X-100 in PBS.

This image is courtesy of an Abreview submitted by Bryan Niedenberger

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)

ICC/IF image of ab6160 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 (ab6160, 2µg/ml) overnight at +4°C. The secondary antibody (green) was ab150165 Alexa Fluor® 488 goat anti-rat IgG (H&L) used at 2µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

• Alexa Fluor®

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Alexa Fluor® - Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (AB150165)