Anti cd34 antibody

Human brain metastasis and glioblastoma biopsies were processed into formalin-fixed and paraffin embedded specimens and sectioned at 6 μm. Tissue sections were deparaffinized and rehydrated, then immunohistochemically stained for VCAM-1 (20 μg/100 uL catalog no. sc8304; Santa Cruz Biotechnology). Adjacent sections were stained for tumor-specific markers: anti-cytokeratin purified clone CAM5.2 (12.5 μg/mL catalog no. 345779; BD Biosciences) for breast cancer and lung adenocarcinoma, and anti-melan A (1:100 catalog no. ab5106; Abcam) for melanoma. Additional sections were stained with anti-CD34 antibody (1:50 catalog no. M7165; Dako) for vascular characterization. In the case of the glioblastoma specimens, insufficient tissue was available for tumor-specific or blood vessel staining. For detailed information on histologic analysis, see Supplementary Materials and Methods.

Consecutive 3 μm sections were cut from each block, and immunohistochemistry was performed as we described previously. An immunoperoxidase technique was done following antigen retrieval with microwave treatment (95°C) in citrate buffer (pH 6.0) for 45 min. After endogenous peroxidase block by 3% H₂O₂-methanol for 15 min, specimens were rinsed with phosphate-buffered saline (PBS) three times. Anti-Prox1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-FOXC2 antibody (Santa Cruz Biotechnology), anti-CD34 antibody (a marker for vascular endothelial cells) (DAKO, Carpinteria, CA, USA), and anti-LYVE-1 antibody (a marker for lymphovascular endothelial cells) (Abcam, Tokyo, Japan) diluted by 0.5 μg/ml were used for primary antibody. After 2 h incubated at room temperature, specimens were rinsed with PBS three times and treated for an hour at room temperature with the secondary antibody peroxidase-conjugated anti-mouse (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan) diluted at 0.5%. The specimens were then rinsed with PBS three times and color-developed with diaminobenzidine (DAB) solution (DAKO). After washing, specimens were counterstained with Meyer’s-hematoxylin (Sigma Chemical Co., St. Louis, MO, USA). Immunostaining of all samples was performed at the same conditions of antibody reaction and DAB exposure.

Paraffin sections from skeletal muscle specimens underwent de-paraffinization and rehydration. Antigen retrieval was performed by microwaving the sections in citrate buffer, pH 6.0. Endogenous peroxidase was blocked with methanol containing 3% hydrogen peroxide. Sections were incubated overnight at 4 °C in humid chamber with the primary antibodies: Anti-CD34 Antibody (Clone: QBEnd-10, Mouse anti-Human, Supplier: DAKO, Catalog Number: M7165), in an optimal dilution of 1:50–1:100, followed by application of secondary antibody (Biotin-streptavidin link, DAKO). Then, the antigen was localized by the addition of 3,3´-diaminobenzidine tetrahydrochloride (DAB) substrate chromogen solution (Universal Detection Kit, DAKO Envision, Denmark). Finally, slides were counterstained with hematoxylin, dehydrated in alcohol and mounted. For each setting, positive and negative control slides were included. As a negative control, skeletal muscle tissue was processed in the above-mentioned sequences, but the primary antibodies were not added and instead add non-immune immunoglobulin G (IgG; DAKO, Glostrup, Copenhagen, Denmark).