Pegfilgrastim was diluted into 0 15 M sodium acetate right befor

Pegfilgrastim was diluted into 0. 15 M sodium acetate right before use. Sodium acetate injections were www.selleckchem.com/products/Dasatinib.html used as a vehicle control. The treatment was started at the age of 12 weeks for survival studies or at 12 16 weeks for histological studies. The treatment was con tinued once a week until the mouse was sacrificed. The littermates were randomly and equally divided into treatment groups. For histological studies, the pegfilgras tim treated mice were sacrificed at the same age as when their littermates reached the clinical end stage. Immunohistochemistry The mice were anesthetized with an overdose of tribro moethanol and transcardially perfused with heparinized saline to remove blood from tissues. The meninges were removed Inhibitors,Modulators,Libraries from spinal cord which was then cut in half at the mid lumbar area and pre pared as paraffin embedded sections and cut with a microtome into 5 um sections.

The spinal cord sections were immunostained with microtubule associated pro tein 2, neuronal nuclear antigen, choline acetyltransferase, glial fibrillary acidic protein and ionized calcium binding adaptor mole cule 1, Inhibitors,Modulators,Libraries followed by the detection with a fluorescent or diaminobenzidine based method as described. The immunoreactive area was quantified from the ventral horn of the spinal cord. The total of 2 mm area of the mid lumbar spinal cord in each mouse was covered, analyzed Inhibitors,Modulators,Libraries as Inhibitors,Modulators,Libraries 20 sections 100 um apart. Blood sample analysis The blood sample was taken from the saphenous vein or from the heart at the time of sacrifice. Repeated blood sampling was avoided to keep the manipulation of hematopoietic system minimal.

The blood sample was mixed with heparin and centrifuged to separate the blood cells. The plasma was collected and stored at 70 C until use. The GCSF concentration in blood was ana lyzed Inhibitors,Modulators,Libraries from plasma samples with a human GCSF ELISA kit according to manufacturers protocol. The flow cytometry staining of blood cells was per formed as described. Briefly, nonspecific antigen binding was blocked with mouse IgG and the cells were stained with fluorochrome conjugated PE CD117, PerCP Gr 1 and FITC CD45, and analyzed on a FACS Calibur equipped with a single 488 nm argon laser. The data are shown as percentage of CD117 or Gr 1 cells of total CD45 blood leukocytes. Cell isolation and cell culture Spinal cord neurons were obtained from E14 mouse embryos with a protocol modified from Vartiainen et al.

Briefly, embryos were decapitated and the spinal cords were isolated. The meninges and the dorsal www.selleckchem.com/products/chir-99021-ct99021-hcl.html root ganglia were removed. Spinal cords were digested in 0. 5 mg ml papain, 0. 04 mg ml DNAse in PBS 5 10 min at 37 C. Papain solution was replaced with 1 mg ml BSA, 0. 04 mg ml DNAse, 10 mM glucose in PBS and gently triturated and centrifuged. The pellet was resus pended into DMEM, 10% FBS, 2 mM glutamine, penicil lin streptomycin and plated at 2. 25 �� 105 cells cm2 onto poly D ornithine coated multiwell plates.

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