重组Anti-Cytokeratin 8抗体[EP1628Y] - Cytoskeleton Marker (ab53280)

Flow cytometry overlay histogram showing left NIH3T3 positive cells and right negative Raw264.7 stained with ab53280 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab53280) (1x 10⁶ in 100μl at 0.2μg/ml (1/11500)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

Lanes 1 - 4: Merged signal (red and green). Green - ab53280 observed at 55 kDa. Red - loading control, ab8245 observed at 37 kDa.  

 ab53280 was shown to react with Cytokeratin 8 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255400 (knockout cell lysate ab263785) was used. Wild-type and Cytokeratin 8 knockout samples were subjected to SDS-PAGE. ab53280 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 (For unpurified use at 1/25,000 - 1/50,000) dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of HT-29 (Human colorectal adenocarcinoma epithelial cell) cells labeling Cytokeratin 9 with Purified ab53280 at 1:500 dilution. Cells were fixed in 100% Methanol and permeabilized with None. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Alexa Fluor^® 488 (ab192467) and Alexa Fluor^® 647 (ab192468) conjugated versions are available for this clone.

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Cytokeratin 8 with purified ab53280 at 1/20 dilution (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluorr^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

Alexa Fluorr^®488 (ab192467) and Alexa Fluorr^®647 (ab192468) conjugated versions are available for this clone. 

Blocking and diluting buffer: 5% NFDM/TBST.

ab53280 (purified) at 1:20 dilution (0.2μg) immunoprecipitating Cytokeratin 8 in HeLa whole cell lysate.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate, 10μg
Lane 2 (+): ab53280 & HeLa whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab53280 in HeLa whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Panx1^-/- mice have normal mammary gland epithelial differentiation at lactation

Immunofluorescent analysis of luminal epithelial marker keratin 8 (green) and myoepithelial marker keratin14 (red) revealed a similar staining pattern in Panx1^-/- mice compared to control mice during lactation. Paraffin-embedded tissue samples.

Hoescht (blue) denotes nuclei. N = 6. Scale bars = 50 um. 

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) labelling Cytokeratin 8 with purified ab53280 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised by 0.1% tritonX-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody (Ab150077). Nuclei counterstained with DAPI (blue).

Unpurified ab53280 (1:250) staining human Cyotkeratin 8 in human breast adenocarcinoma tissue by immunohistochemistry using paraffin embedded tissue.

Unpurified ab53280 staining Cytokeratin 8 in Mouse liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formalin and blocked with 10% serum for 20 minutes at 23°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/75 in TBS + 1% BSA) for 1 hour at 23°C. A Biotin-conjugated Goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

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Immunofluorescent staining of HeLa cells using unpurified ab53280 (1:100).

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 RT Incubate in 7.5% sucrose-PBS for 3h at RT Incubate in 15% sucrose-PBS at 4 degree Celsius overnight Embed the EBs in tissue-Tek OCT compound Cut frozen sections to 4-20 µm thickness

Primary antibody 1: Rabbit anti cytokeratin 8 (unpurified ab53280), 1:100

Primary antibody 2: Rat anti-perlecan, 1:100
Secondary antibody 1: Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488) pre-adsorbed (ab150081), 1:200

Secondary antibody 2: Goat polyclonal Secondary Antibody to Rat IgG - H&L (Cy5®) pre-adsorbed (ab150081), 1:200
Nuclei were counterstained with DAPI

Fluorescent immunohistochemical analysis of paraffin-embedded human ovarian carcinoma tissue using unpurified ab53280. Green-CK8 red-PI.

Fluorescent immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using unpurified ab53280. Green-CK8 red-PI

Fluorescent immunohistochemical analysis of paraffin-embedded human colonic adenocarcinoma tissue using unpurified ab53280. Green-CK8 red-PI.

Fluorescent immunohistochemical analysis of paraffin-embedded human endometrial carcinoma tissue using unpurified ab53280. Green-CK8 red-PI.

Overlay histogram showingHeLa cells stained with unpurifiedab53280 (red line). The cells were fixed with 2%PFA (room temperature, 30 min) and then permeabilized with 1%FACS permeabilizing solutionfor 30 min. The cells were then incubated in3% FBS in 1X PBSfollowed by the antibody (ab53280, 1/20 dilution) for1 hourat room temperature. The cells were then incubated for 30 min at room temperature with the secondary antibody. An isotype control antibody (black line) was used and an unlabelled sample (blue line) was also used as a control.