While the effect of these antibodies on IL 21 PK was not analyzed, the developm

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While the effect of these antibodies on IL 21 PK was not analyzed, the developm

Post  huwan123456 on Tue May 20, 2014 4:56 am

Generation of immune complexes Mice were anaesthetized by intraperitoneal injection of 0. 1 ml 5 g body weight Avertin . The scalp was shaved and local anaesthetic was placed in the external auditory meati as mice were positioned in a stereotaxic frame . 10 mg of OVA was freshly diluted in 1 ml of saline, and 1ul injected into the stri atum of OVA immunized or control non [You must be registered and logged in to see this link.] immunized mice. Injections were performed over a period of 2 mi nutes through a glass micropipette with an injecting tip of 50 um . Tissue was col lected at 5 minutes, 3 h, 24 h, or 7 days after the injections. For analysis of immune complex formation, mice were perfused with heparinized saline and brains immediately frozen in Tissue Tek OCT . Blood samples were taken to assess OVA specific antibody titers by ELISA and described before .

The [You must be registered and logged in to see this link.] brains were trimmed to isolate coronal blocks of 7 mm thickness that contained the injection site in the centre and cut into 10 um thick coronal sections on a cryostat. Sections were collected on APES coated slides for histological examination, quantification studies and immunocytochemistry. Sections of brain were stained by immunohistochem istry to identify elements characteristic of immune com plexes and to study their position in relation to cerebral vascular basement membranes. Complement was iden tified by antibodies against mouse C3, at a dilution of 1,2000. Mouse IgG was identified using FITC labelled F 2 fragments of goat anti mouse IgG, at 1,500 dilution. Rabbit anti OVA was used at a dilution of 1,1000. Activated macrophages were studied using an F4 80 monoclonal antibody, at 1,500 dilution.

To assess the distribution of the immune complexes in relation to the cerebrovascular basement membranes, simultaneous staining of the laminin component of vas cular basement membranes was [You must be registered and logged in to see this link.] performed by immuno cytochemistry using a pan laminin polyclonal rabbit antibody at a 1,500 dilu tion. Smooth muscle cells of the tunica media of arteries were identified by detecting smooth muscle actin using a mouse monoclonal antibody at 1,4000 dilution. Injections of fluorescent tracer At 5 minutes, 24hr and 7 days following the intracere bral injections of OVA, fluorescein labeled 3 kDa soluble lysine fixable dextran was injected into the striatum, as described previously . Dextran was injected at 1 ug ul in a volume of 0.

5 ul, over a period of 2 minutes through a glass micro pipette with the injecting tip 50 um as previously described . Mice were sacrificed at 5 minutes following injection of the tracer. Tissue was collected after terminal anaesthesia with sodium pentobarbitone and transcardially perfusion with heparinized 0. 9% saline followed by 4% paraformaldehyde in 0. 1 M phosphate buffer pH 7. 4. Brains were removed and further fixed by immersion in 4% paraformaldehyde for 4 6 hours and placed in 30% sucrose for 48 h for cryoprotection. The brains were trimmed to form coronal blocks 7 mm thick with the injection site in the centre. Blocks were then fro zen in Tissue Tek OCT and sectioned in a coronal plane on a cryostat. Sections were collected on gelatin coated slides for histological examination, quan tification studies and immunocytochemistry.


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