Preparation of brain tissue and immunoprecipitation experiments were performed as previously described [ 5] without antibodies as a negative control, with 1 15 μg mMLC1-C (home made), with the nonspecific CHOP (Santa Cruz, Heidelberg, Germany) as an extra negative control, and with Kir4.1 (USBiological, Swampscott, Massachusetts) antibodies.
If brain tissue is available, it is sent to Dr. Gambetti, who can confirm the diagnosis.
For experiments using dissected brain tissue, 20 brains were dissected per sample and cRNA probes were made by converting the RNA to double stranded cDNA that is flanked by a T7 promoter.
Preadsorption experiments on H. assulta brain tissue were carried out by applying a conjugate control for serotonin, that is, lyophilized serotonin creatine sulfate coupled to bovine serum albumin (Immunostar, Hudson, WI) at a concentration of 20 μg/mL.
In all the experiments samples of brain tissue were microdissected [ 19] from areas of the brain (hippocampus and cerebellum) of groups of age matched (~10 weeks old) animals injected intracerebrally with either TSE (scrapie isolate, ME7) infected or normal brain homogenate inocula.
The brain tissues were harvested for next experiments, and were used for the measurement of brain infarction (n = 6 per a group), edema (n = 3 per a group), morphological changes of neuronal cells (n = 3 per group), Western blot (n = 3 per group), and BBB permeability (n = 3 per group).
The lesion volume and the amount of histologically intact residual brain tissue were measured at the end of the experiment.
Although brain tissue would be the optimal choice, its use in MD transcriptome experiments is challenging.
All experiments were performed on brain tissue from MOP/NOP KO animals.
For this a series of optimisation experiments were performed on brain tissue samples (no. = 4 ME7 terminal 4 NB).
