Immunocytochemistry/ Immunofluorescence analysis of JAR (Human placenta choriocarcinoma cell line) cells labeling Ubiquitin with Purified ab134953 at 1:100 dilution (7.2μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. 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.

Intracellular Flow Cytometry analysis of JAR (Human placenta choriocarcinoma cell line) cells labeling Ubiquitin with purified ab134953 at 1/70 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 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). 

Blocking and diluting buffer: 5% NFDM/TBST

ab134953 staining Ubiquitin in Mouse embryo tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% FBS/BSA for 1 hour at room temperature; antigen retrieval was by heat mediation in citrate buffer, pH6. Samples were incubated with primary antibody (1/100 in 1% FBS/BSA) for 16 hours at 4°C. An undiluted HRP-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat liver tissue sections labeling Ubiquitin with Purified ab134953 at 1:800 dilution (0.9 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse liver tissue sections labeling Ubiquitin with Purified ab134953 at 1:800 dilution (0.9 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP)
secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder carcinoma tissue sections labeling Ubiquitin with Purified ab134953 at 1:800 dilution (0.9 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, pH 9.0. Tissue was counterstained with Hematoxylin. ab97051 Goat Anti-Rabbit IgG H&L (HRP) secondary antibody was used at 1:500 dilution. PBS instead of the primary antibody was used as the negative control.

Blocking and diluting buffer: 5% NFDM/TBST

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes .ab134953 and ab8245 (loading control to GAPDH) were diluted 1/200  and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging using the Licor Odyssey CLx.