Mouse brain inoculation for cats. Protocol: MJerome060601 Ó2001 Michael White-Veterinary Molecular Biology || Questions at mjerome@montana.edu
WhiteProtocols2001
1.0 Mouse Brain Inoculation for Cats. 1.1 In a 15 ml screw-cap tube, wash stored oocysts with 10 ml Hanks (pH 7.2) (pellet 10 min at 1300 rpm in a Sorvall RT6000B) to remove sulfuric acid (pellet 10 min at 1300 rpm in a Sorvall RT6000B). Repeat (Hanks will turn a bright yellow until the sulfuric acid is gone). 1.2 Count oocysts and dilute to 5,000 per ml. Inoculate CD-1 mice sub-cutaneously with 200 ml, and house for 1 month. 1.3 At 1 month, euthanize the mice. With a sharp pair of scissors, decapitate the mice. Make a longitudinal cut through the skin and then through the skull. Make 2 transverse cuts behind the eye sockets that intersect the first cut. Fold back the skullcap and extract the whole brain using a pair of forceps. 1.4 Place the brain into a tissue grinder and add 1 ml of 0.25% Trypsin in PBS. Slowly homogenize the brain. Pipet the homogenate into a 1.5 ml tube. Wash the grinder with 0.5 ml of 0.5% trypsin and pipet into the same tube. At this point a small sample may be taken to look for brain cysts under a microscope. 1.5 Place the tube into a 37^oC water bath for 30 min. 1.6 Spin down homogenate for 5 min in a micro centrifuge and wash with 10% FBS media to remove trypsin. Bring up pellet in 4 ml Hanks (pH 7.2). The resuspended pellet will appear thick and viscous. Again CD-1 mice sub-cutaneously using 200 ml of the suspension. 1.7 House the mice for 2 months before inoculating cats. Remove the brains as described. Cats should be 8-9 weeks old at the time of inoculation. Feed 1 brain per cat.
Department of Veterinary Molecular Biology, Montana State University, Bozeman MT