Donkey Anti-Mouse IgG H&L (Alexa Fluor® 594)

• ICC/IF

AbReview41355****

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Mouse IgG H&L (Alexa Fluor® 594) (AB150108)

ab16287 staining SSEA4 in rat stem cells derived from tendon by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with parafolmaldehyde, permeabilized with PBS containing 0.25% Triton X-100 and blocked with 1% BSA for 30 minutes at room temperature. Samples were incubated with primary antibody (1/100) for 20 hour at 4°C. An Alexa Fluor® 594-conjugated donkey anti-mouse IgG H&L (ab150108) (1/1000) was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Sun Jeong Kim.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Mouse IgG H&L (Alexa Fluor® 594) (AB150108)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized HAP1 WT and HAP1-CANX KO cells labelling Calnexin with ab328433 at 1.0 μg/ml dilution, followed by ab150133 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Left four panels : Goat chimeric ab328433 staining CANX [EPR3632] in HAP1 (positive) and HAP1-CANX knockout (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 for 2 hours at room temperature with ab328433 at 1 ug/ml (shown in green) and ab7291, Anti-alpha Tubulin antibody [DM1A] - Loading Control (shown in pseudocolour magenta). Cells were then incubated with ab150133, Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution and ab150108, Donkey Anti-Mouse IgG H&L (Alexa Fluor® 594) 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, Revvity) and a maximum intensity projection of confocal sections is shown.

Right four panels : Side-by-side testing with the original rabbit version of the same clone, ab92573 (Anti-Calnexin antibody [EPR3632].)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Mouse IgG H&L (Alexa Fluor® 594) (AB150108)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Hela treated with 2µM Straurosporine for 24 hours and untreated HeLa cells labelling Cleaved Caspase-3 with ab328434 at 5.0 μg/ml dilution, followed by ab150133 Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Left four panels : Goat chimeric ab328434 [E83-77] staining Cleaved Caspase-3 in HeLa cells treated with 2µM Straurosporine for 24 hours to induce cell death (positive) and untreated HeLa cells (negative). 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 donkey serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated for 2 hours at room temperature with ab328434 at 5 µg/ml (shown in green) and ab7291, Anti-alpha Tubulin antibody [DM1A] - Loading Control (shown in pseudocolour magenta). Cells were then incubated with ab150133, Donkey Anti-Goat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution and ab150108, Donkey Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed at 1/1000 dilution. Nuclear DNA was labeled with DAPI (shown in blue). Also suitable in cells fixed with 100% methanol (5 min). Image was acquired with a high-content analyser (Operetta CLS, Revvity) and a maximum intensity projection of confocal sections is shown.

Right four panels : Side-by-side testing with the original rabbit version of the same clone, ab32042 (Anti-Cleaved Caspase-3 antibody [E83-77]).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Donkey Anti-Mouse IgG H&L (Alexa Fluor® 594) (AB150108)

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 donkey 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, 2μg/ml) overnight at +4°C. The secondary antibody (orange) was ab150108 Alexa Fluor® 594 donkey anti-mouse IgG (H+L) used at 1μg/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.

• Alexa Fluor®

Unknown

Alexa Fluor® - Donkey Anti-Mouse IgG H&L (Alexa Fluor® 594) (AB150108)

Emmission spectra of the Alexa Fluor^® conjugated secondary antibodies.