Biotin conjugated anti mouse igg

The technique employed was adapted from Mebs^{20 (link)}. Paraffin sections from four randomly chosen specimens of each population were treated with 0.3% hydrogen peroxide and 20% bovine serum albumin and incubated with commercial monoclonal anti-TTX antibody (1:15000 for LW⁻ and 1:60000 for SC⁺; Hawaii Biotech) followed by biotin-conjugated anti-mouse IgG (Dako®). As an enzyme marker, the avidin and biotinylated horse radish peroxidase macromolecular complex kit (Dako®) was used, and distribution of the antigen in skin was visualized by 3,3′-diaminobenzidine (DAB) substrate solution for 30 sec. Sections were counterstained with hematoxylin. Positive stain of the antigen was recognized as a goldish or brownish color. For controls, we employed the same method described above using 0.2% bovine serum albumin instead of the anti-TTX antibody.

A Mk colony formation assay was performed as described previously^{33 (link),34 (link)} with minor modifications. CD34-positive cells were purified from cryopreserved bone marrow mononuclear cells (BM-MNCs) using CD34 MicroBead Kit UltraPure (MACS Miltenyi Biotec). CD34-positive cells (5000) were cultured in a chamber (two-chamber slide, Matsunami) with serum-free collagen medium in the presence of 20 ng/mL human IL-3 (hIL-3) (Miltenyi Biotec) and human IL-6 (hIL-6) (Peprotech) and in the presence or absence of 50 ng/mL human thrombopoietin (hTPO) (Kyowa Kirin). The cultures were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air for 12–14 days. Fixed cells were stained with anti-CD41 antibody (Clone HIP8, STEMCELL), biotin-conjugated anti-mouse IgG (Dako), streptavidin-conjugated alkaline phosphatase (Vector Laboratory), SIGMA FAST Red TR/naphthol AS-MX alkaline phosphatase substrate (Sigma), and Evans Blue (FUJIFILM Wako). BM-MNCs collected from lymphoma patients without BM infiltration and reactive thrombocytosis patients were used as negative controls.