Recombinant Anti-Hes1 antibody [EPR4226] - BSA and Azide free (ab221788)

Immunofluorescent analysis of 4% Paraformaldehyde fixed 0.1% TritonX-100 permeabilised SH-SY5Y (Human neuroblastoma epithelial cell) labelling Hes1 with ab108937 at 1/1000 dilution, followed by AlexaFluor^® 488 Goat anti-Rabbit secondary (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in SH-SY5Y cell line  is observed. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)  (ab195889) was used to counterstain alpha Tubulin  at 1/200 dilution (red). The Nuclear counterstain was DAPI (blue).

Secondary antibody only control: Secondary antibody is AlexaFluor® 488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

This data was developed using ab108937, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration: 5% NFDM /TBST.

This data was developed using ab108937, the same antibody clone in a different buffer formulation.

Primary ab concentration dilution: 1:200, (0.5ug/ml), Secondary ab: ImmunoHistoprobe (Ready to use) HRP Polymer for Rabbit IgG, Secondary ab concentration: Prediluted, Tissue: Human brain, Fixative: Paraffin-embedded sections, Counter stain: Hematoxylin Antigen retrieval: Perform heat mediated antigen retrieval using Tris/EDTA Buffer, PH9

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108937).

Clone EPR4226 (ab221788) has been successfully conjugated by Abcam. This image was generated using Anti-Hes1 antibody [EPR4226] (Alexa Fluor® 488). Please refer to ab196328 for protocol details.

ab196328 staining Hes1 in SKNSH cells. The cells were fixed with 100% methanol (5min), 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 overnight at +4°C with ab196328 at 1/100 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 2 µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Clone EPR4226 (ab221788) has been successfully conjugated by Abcam. This image was generated using Anti-Hes1 antibody [EPR4226] (PE). Please refer to ab225087 for protocol details. 

Overlay histogram showing SHSY5Y cells stained with ab225087 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab225087, 1/1000 dilution) for 30 min at 22°C. 

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control. 

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter. 

Immunohistochemical staining of Hes1 in paraffin-embedded human placenta tissue with unpurified ab108937 at 1/250 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108937).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemical staining of paraffin embedded human brain with unpurified ab108937 at a working dilution of 1 in 90. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108937).

Clone EPR4226 (ab221788) has been successfully conjugated by Abcam. This image was generated using Anti-Hes1 antibody [EPR4226] (Alexa Fluor® 647). Please refer to ab196577 for protocol details.

ab196577 staining Hes1 in SK-N-SH cells. 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 overnight at +4°C with ab196577 at 1/200 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
This product also gave a positive signal under the same testing conditions in SK-N-SH cells fixed with 100% methanol (5min).

Overlay histogram showing SH-SY5Y cells stained with unpurifiedab108937 (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. The cells were then incubated with the antibody (ab108937, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108937).