Anti-SC35 antibody [SC-35] - Nuclear Speckle Marker (ab11826)

Immunocytochemistry/ Immunofluorescence analysis of HEK-293T and RKO cells transiently transfected with CDX2/AS-His and co-stained for CDX2/AS-His and SC35 (ab11826). All proteins localized to the nucleus and merged images revealed co-localization of CDX2/AS with SC35.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human hippocampus tissue sections labeling SC35 with ab11826 at 1/500 dilution. The tissue was fixed with formaldehyde and a heat mediated antigen retrival step was performed with a pH 9 buffer. Blocking of the sample was done with 100% PBS for 1 hour at 25°C, followed by staining with ab11826 at 1/500 in PBS for 14h at 4°C. A biotinylated goat anti-mouse polyclonal antibody at 1/200 was used as the secondary antibody.  

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Immunocytochemistry/ Immunofluorescence analysis of HEK-293 human kidney cells labeling SC35 with ab11826 at 1/400 dilution. Cells were fixed with methanol and blocked with PBS for 1 hour at 4°C. Staining with ab11826 was carried out in PBS buffer for 2 hours at 4°C. An undiluted goat anti-mouse Alexa Fluor^® 594 secondary antibody was used. 

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Immunocytochemistry/ Immunofluorescence analysis of untransfected U-2 OS cells (A) and cells transfected with HSV US1 or US1.5 fixed and stained for FLAG (red) and SC35 (green) to identify viral proteins and nuclear speckles respectively. Transfected cells were fixed 40 h post transfection with 3.7% formaldehyde in PBS (20 min), permeabilized with 0.5% Triton X-100 in PBS (10 min), and blocked with 4% BSA in PBS (20 min) prior to incubation with Anti-SC35 antibody [SC-35] - Nuclear Speckle Marker (ab11826) and secondary antibodies in 4% BSA in PBS. DAPI was used for visualization of nuclear DNA. Scale bar  =  10 µm.

Immunocytochemistry/ Immunofluorescence analysis of human adenocarcinoma HT-29 (Human colorectal adenocarcinoma cell line) cells labeling SC35 with ab11826 at 1/200 dilution. Cells were fixed in paraformaldehyde and permeabilized with Triton X-100 and Saponin. Blocking of the cells was done with 1% BSA for 1 hour at 37°C; staining with ab11826 at 1/200 was carried out for 16 hours at 4°C in PBS buffer. An anti-mouse IgG3 (Alexa Fluor^® 594) secondary antibody was used at 1/200 dilution. 

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Immunocytochemistry/ Immunofluorescence analysis of  human hippocampus cells labeling SC35 with ab11826 at 1/200 dilution. Cells were fixed with formaldehyde and blocked with PBS for 1 hour at 4°C. Staining with ab11826 was carried out in PBS buffer for 12 hours at 4°C. A goat anti-mouse Alexa Fluor^® 594 secondary antibody was used at 1/1000 dilution. 

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Immunohistochemistry (Frozen Sections) analysis of human retinal sections (16 µm cryosections) labeling SC35 with ab11826 at 1/300 dilution (red).

The cryosections were first hydrated in phosphate-buffered saline (PBS, pH 7.4) and washed three times (5 minutes each at room temperature (RT)), followed by incubation with PBTS buffer (1X PBS with 0.1% triton-X 100, 0.2% BSA and 0.02% SDS) for an hour at RT. Primary antibody Anti-SC35 antibody [SC-35] - Nuclear Speckle Marker (ab11826) was incubated in PBTS buffer overnight at 4°C. Sections were washed with PBTS buffer containing DAPI 10 times (15 minutes each at RT). Following the washes, secondary antibody anti-mouse antibody conjugated with Alexa Fluor^® at 1/750 dilution was incubated in PBTS buffer overnight at 4°C. Sections were washed with PBTS buffer 7 times (15 minutes each at RT), rinsed with PBS and covered with anti-fade reagent and coverslip glass.

ab11826 staining SC35 in human fibroblast cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.3% Triton X-100 in PBS and blocked with 5% Normal Goat Serum/0.3% Triton X-100 in PBS for 60 minutes at 25°C. Samples were incubated with primary antibody (1/500 in 1% BSA/ 0.3% Triton X-100 in PBS) for 16 hours at 4°C. An Alexa Flour^® 488 goat anti-mouse IgG (H+L), F(ab')2 Fragment Ig was used as the secondary antibody at a dilution of 1/1000.

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ab11826 (1/1000) staining SC35 (phospho) in human retinal pigment epithelial (RPE) cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (blue). Please refer to abreview for further experimental details.

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ab11826 staining cultured human colon adenocarcinoma HT-29 cells.

Cells were PFA fixed and permeabilized in Triton X-100 and saponin prior to blocking with 1% BSA for 1 hour at RT. The primary antibody was diluted 1/200 and incubated with the sample for 16 hours at 4°C. An Alexa Fluor^® 594 conjugated goat anti-mouse IgG3 antibody was used as the secondary.

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HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were fixed 24–48 hours after transfection using 4% paraformaldehyde, permeabilized with 0.2% triton X-100/PBS and probed with ab11826 followed by FITC conjugated secondary antibodies (green). After washing with PBS, slides were mounted using Citifluor and analysed by confocal microscopy. Cells were visualized under a Leica laser scanning confocal microscope equipped with a DM-RXE microscope and an argon-krypton laser.

ICC/IF image of ab11826 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells. The cells were fixed in 100% methanol (5 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab11826 at 1 µg/ml overnight at +4°C. The secondary antibody (green) was DyLight^® 488 goat anti- mouse (ab96879) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.