Rabbit polyclonal cleaved caspase 3 antibody

Mouse monoclonal and rabbit polyclonal HA, mouse monoclonal GFP and rabbit polyclonal cdk2 antibodies were from Santa Cruz Biotechnology. Rabbit polyclonal cleaved caspase-3 antibody was from Cell Signaling Technology. Mouse monoclonal TFR antibody and rabbit polyclonal TBC1D17 antibody were obtained from Invitrogen. Rabbit polyclonal p62/SQSTM1 antibody was from Sigma-Aldrich and mouse monoclonal LC3 antibody was from Enzo Life Sciences. Secondary antibodies Cy-3 conjugated anti-mouse and anti-rabbit IgG, and HRP conjugated anti-mouse and anti-rabbit IgGs were from Amersham. Alexa-488 and Alexa-633 conjugated anti-mouse and anti-rabbit IgGs were from Molecular Probes.

Tumors of OPM-2 CRBN KO (CRBN knockout cells) were isolated when the SCID mice were sacrificed 13 days after starting the treatment with the vehicle or CUDC-907 (the next day after all oral administrations are completed). Formalin-fixed paraffin-embedded sections (3 μm) were deparaffinized and hydrated with graded ethanol. After heat-induced antigen retrieval for 10 min at 120 °C, slides were incubated in the following primary antibodies: a 1:300 dilution of cleaved caspase-3 rabbit polyclonal antibody (Cell Signaling Technology, #9661, Massachusetts, USA), a 1:200 dilution of GSK3 beta (phospho Y216) + GSK3 alpha (phospho Y279) rabbit polyclonal antibody (Abcam, ab75745, Cambridge, UK), a 1:100 dilution of c-Myc rabbit monoclonal antibody (clone Y69, Abcam, ab32072) for 30 min at 37 °C respectively. Labeling of secondary antibody was performed with Histofine Simple Stain Mouse MAX-PO(R) (Nichirei Bioscience, Tokyo, Japan) for 30 min at 37 °C. To visualize the antigen-antibody complex, the ImmPACT DAB substrate kit (Vector Laboratories, Burlingame, CA, USA) was used, and then sections were counterstained with hematoxylin. A section from a breast cancer tissue placed on the same slide glass was used as the positive control, and sections treated with PBS instead of the primary antibodies were used as negative controls.

Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of alcohol. The antigen was retrieved with 0.01 M citrate buffer (pH 6.0) by heating the sample in microwave for 10 min. The tissue sections were then placed in 3% hydrogen peroxide for 5 min to inactivate the endogenous peroxidase, and blocked for 30 min with normal horse serum (DakoCytomation LSAB2 System-HRP kit; DakoCytomation, Glostrup, Denmark). The primary antibodies used for this study were Ki-67 rabbit polyclonal antibody (1:300; Abcam, Cambridge, MA, USA), VEGF and SIRT1 mouse polyclonal antibodies (1:150; all from Abcam), cleaved caspase-3 rabbit polyclonal antibody (1:300; Cell Signaling), and B-cell lymphoma-extra large (Bcl-xL) mouse monoclonal antibodies (1:100: Santa Cruz biotechnology, Dallas, TX, USA). The prediluted primary antibodies were applied overnight at 4 °C. The slides were then treated with biotinylated secondary antibody for 30 min at room temperature, followed by the treatment with streptavidin-HRP and 3,3′-diaminobenzidine solution for another 10 min at room temperature. Tissue sections were counterstained with hematoxylin.

For detection of Hoxd10 protein, polyclonal antibodies were raised in rabbits using the peptide sequence, SQVESPEAKGGLPEDR, and verified by Western blot (Covance). Two antisera, designated 1159 and 1160 (1:200 dilution), produced qualitatively similar results. Tissue sections (3 μm) were subjected to antigen retrieval under Tris-HCl buffer, 0.1 M, pH 9.0 by heating to 120 °C for 10′ in a pressure cooker. Primary antibodies used include: rabbit anti-mouse β-casein [56 (link)], Ki67 (MIB-1) monoclonal antibody (Dako), Cleaved caspase-3 rabbit polyclonal antibody (Cell Signaling Technology), STAT5a rabbit polyclonal antibody (Santa Cruz Biotechnology), pSTAT5 (Tyr694) Rabbit monoclonal antibody (Cell Signaling Technology), pSTAT3 (Tyr705)(D3A7) Rabbit monoclonal antibody (Cell Signaling Technology), and GLUT1 rabbit polyclonal antibody (Alpha Diagnostic International). Secondary antibodies (DakoCytomation) were conjugated to horseradish peroxidase. Detections were performed using the Vectastain Elite system.

Mammary glands or mammary tumors were homogenized in RIPA buffer, and the protein extracts were analyzed by Western blot analysis. The cleaved caspase-3 rabbit polyclonal antibody was from Cell Signaling Technology (Cat. No. 9661). The Bcl-2 rabbit monoclonal antibody was from Cell Signaling Technology (Cat. No. 2870). The Smad3 rabbit monoclonal antibody was from Abcam (Cat. No. 40854). The Actin mouse monoclonal antibody was from Sigma (Cat No. A1978).