Biotin conjugated anti rabbit igg

Rats were anaesthetized and transcardially perfused with ice cold phosphate buffered saline (PBS), followed by 4% buffered paraformaldehyde saline (pH 7.4) for in situ fixation of the brain. Brains were isolated, dehydrated, and embedded in paraffin and a series of 5 μm sections were made using microtome (Leica, Germany). After hydration the sections were washed with Tris-buffered saline (TBS, pH 7.4) followed by incubation with proteinase K for 20 minutes for antigen retrieval. After washing with TBS, the sections were incubated with blocking buffer (5% normal goat serum in PBS) for 2 hours. Endogenous biotin binding sites were blocked by sequential incubation of avidin and biotin for 30 minutes each. The sections were then incubated with primary antibody (for tyrosine hydroxylase, Sigma) in blocking buffer at 4°C overnight. The sections were washed three times with PBS and then incubated with biotin-conjugated anti-rabbit IgG (Sigma-Aldrich Inc., USA) in blocking buffer for 2 hours. The specific labelling was detected using diaminobenzidine (DAB). The sections were counterstained with hematoxylin and observed under light microscope (Leica, Germany) and images were acquired with the CCD camera (Leica, Germany) [30 (link)]. The entire study was carried out in a double blind manner.

Dasatinib and nilotinib for in vitro experiments were from Cayman Chemical (Ann Arbor, MI, USA). The following directly conjugated monoclonal antibodies were purchased from Becton Dickinson (San Jose, CA, USA): annexin V-FITC, CD41a-PECy5, CD42a-FITC, CD62P-PE, PAC1-FITC. CD41-PE was from DAKO (Glostrup, Denmark). Dimethylsulfoxide (DMSO), Sepharose CL-2B, anti-actin and biotin conjugated anti-rabbit IgG were from Sigma-Aldrich (St. Louis, MO, USA) and convulxin were from Pentapharm (Basel, Switzerland). For western blot we used the following polyclonal antibodies: p-Fyn Y530, p-Lyn Y396, p-Src Y529, and p-Src Y418 (Thermo Fischer Scientific, Rockford, IL, USA); p-Fyn Y416 and p-Lyn Y507 Biorbyt (San Francisco, CA, USA); and Cell Signaling Technology (Leiden, The Netherlands), respectively. Avidin-biotin complex kit was from Vector Laboratories (Burlingame, CA, USA), and ECL reagent was used from Millipore (Billerica, MA, USA).