Bitan:mouse sac and tissue collection
1/8/10 Aida Attar Sac and Tissue Collection
DAY BEFORE SAC
I) Make sac list: mouse id, DOB, diet, experiment start date, sac date, weight, comments
II) Get tube racks (# of tubes: 7 or 8 brain regions/mouse, 1 tube for plasma, 1 tube for spleen, 1 tube for liver, 1 tube for ear snip for genotyping) A) Label all 1.5ml tubes with animal id and tissue id B) Put plasma tubes on one rack to go by centrifuge C) Put liver and spleen tubes in one rack to go in hood D) Put all tubes for brains of mice on one rack (iow: all 8 of mouse #3’s tubes on one rack) E) Label 15ml tubes for each mouse for 4% paraformaldehyde, could fill with ~2ml and keep in fridge.
III) Make 1x saline with protease and phosphatase inhibitors
IV) Get materials together: A) Nembutal, insuling syringes, scale, 1ml syringe and 21G needle for blood, capiject tubes for blood B) Saline, protease and phosphatase inhibitors: C) Needles and Styrofoam to pin mouse down in a blood collection tub in hood, perfusion machine, tubes and perfusion needle, paper towels D) Forcepts, scissors, straight clamp, curved clamp E) Centrifuge, 100ul pipette, pipette tips, F) Dry ice or liquid nitrogen, body bags G) Large scissors, small scissors, long spatula H) Petri dish on ice in Styrofoam box I) 2 non-ridged pointy forceps and 2 spatulas, scalpal blade, razor J) 4% para, PBS, 30% EtOH
V) DAY of Sacrifice
A) Get mice and bring to Reed B floor sac room B) Give .1cc lethal dose of Nembutal/Pentobarbital IP to mouse (insulin syringes) C) Once mouse is down, weigh it and write on sac list (scale) D) Check for depth of anesthesia by pinching toes with the foreceps, if no flinch is observed, then can use needles to pin spread mouse by arms and legs to the Styrofoam in the blood collection tub in the hood. E) Use forceps to hold skin under the ribs up away from organs, cut with scissors and cut up through diaphram and make a flap F) Keep flap open with straight clamp G) Remove pericardium tissue from around heart with foreceps H) Use forceps to hold heart and put in green/21G needles on 1ml syringe into right ventricle with needle hole towards the middle of the heart and pull on syringe plunger slowly while wiggeling needle a bit to make sure the tissue isn’t suctioned into hole (but if needle hole gets exposed to air, the blot will clot). On a good day, I collect about .6cc of blood. Once you spin, you get about half as much plasma, so try for at least .2cc of blood. 1) When you open the chest cavity, the heart should be pumping relatively quickly to get a good amount of blood. If wait too long after deep anesthesia has been attained, the blood will be beating slower and you will collect less blood. 2) Be careful when you put the needle in, if the needle comes out, the blood may squirt on you I) Once no more blood is coming into needle, pull syringe out of hear, pull needle off, and squirt blood into capiject (lithium/heparin gel barrier: ) tubes. Invert tube to make sure all heparin gets into blood and nothing clots. Take over to centrifuge right away and spin for at least 5min at 10K and at 4degC. If can’t do right away, keep on ice. After spinning, transfer from capiject to 1.5ml labeled tube (100ul pipette and tips). Put plasma in liquid nitrogen or on dry ice to freeze. J) Use curved clamp with right hand to hold heart, insert perfusion needle into left ventricle tip, close clamp and turn on perfusion machine connected to saline+Protease and phosphatase inhibitors. The atriums will swell, right away, cut right atrium, otherwise body will swell. 1) On a good day, the liver will get yellow within less than 20 sec. If this doesn’t happen, might leave on for a while longer, maybe the clamp is blocking the flow of the needle, readjust clamp, OFTEN there is a hole in the heart (possibly the wall between the two ventricles) that the saline is leaking from before being pushed through the body. There’s not much you can do, sometimes I’ll try to bury the heart so the hole is less open and maybe some perfusion happens. K) Once perfusion is complete, cut off a piece of liver and the spleen and put in labeled tubes to keep for toxicity testing. Freeze on dry ice. L) take off clamps and take out perfusion needle, remove needles on hands and feet and put mouse on a paper towel to move to bench top. M) Flip mouse on stomach and use a large scissors to cut head off. Push shoulders down to get a good location for scissors. Place body in double bagged body bags. N) Take out pump from under skin with scissors and forceps and just blunt needle and syringe to check to see if any liquid left in pump. Record result. O) Take ear snip for genotyping.
VI) Brain collection
A) Use large scissors to cut a line from nape of neck to between the eyes in the skin, then use scissors to cut the muscles and extra tissue off around the neck, spread skin apart to see skull B) Use small scissors, put in at spinal cord hole and cut straight along bone joints up to half circle between eyes C) Use spatula’s long side to place parallel to bone cut and flip bone up to remove. This part is tricky, often the spatula will slip and push into the brain. Be careful. Hold head sideways so first bone flip will be the side farther from you, this will give you a better angle for flipping the side towards you. D) Then you’ll need to break the bone between the cortex and the cerebellum AND around the spinal cord to easily get the brain out. Use spatula for this. Then use the spatula to sweep around the brain and cut all the nerves and separate the brain from the skull. E) Flip the brain onto the slightly moist Petri dish (brain cutting station) on ice at the top edge of a styrafoam box.
VII) Brain dissecting and fixation
A) Hold the brain with non-ridged foreceps, use either a scalpal blade or a razor blade to cut cerebellum #8 off. 1) MAKE SURE all cuts are made so they are straight both vertically and horizontally B) Then cut the hemispheres into 2 along the crease. Use a spatula to slide the left half off the razor as the RIGHT half is still stuck to the razor, pick it up and put it in the Formalin jar. Don’t touch the jar with the razor or get formalin on the razor because then it will fix the left half of the brain when cutting 1) The right half brain needs to be in the 4% para for ~10hrs (1mm/hr of penetration at room temp). I put them at room temp all of sac day then put them in the fridge over night (6hrs Rm/T + 18hrs 4degC for 10cm thickness) 2) Then wash 1hr in PBS, (I did overnight) 3) Then into 30% EtOH at 4degC or 70% EtOH in -20degC. Can be stored for several weeks before embedding. C) On the left brain half, make one cut just anterior to the corpus callosum this is the frontal #1 and septum and olfac tract#2. Use two spatulas to separate the olfactory tract, then separate the cortex (outer) from the septum (inner). D) Then cut almost in middle of rest of brain, just anterior to the notch on the bottom. 1) When razor is between the 2 brain pieces, use a spatula to push flat the 2 halves (2 non-ridges foreceps work too) such that the inner part is up. If cut correctly, the anterior half hippo will NOT be extended all the way down but the post half hippo will be. If both sides look the same with a tail/extension down, then cut too post and this affects #3 and #7. E) Make two cuts to take out the southeast quadrent of the post half #7 (entorhinal cortex) and the southwest quadrent of the ant half #3 (piriform cortex). They should be about the same size F) Then use 2 spatulas to push apart the thalamus, the hippo, and the cortex. Push thalamus down, push cortex up, then peal the hippo out. Post hippo is bigger than ant hippo. Put sections from both halves together. 1) Cortex #4, hippo #5, thalamus #6 G) Throw tubes into liquid nitrogen to snap freeze. Dry ice seems to work too. H) Record time for measuring formalin penetration.
1 frontal cortex 2 olfact and septum 3 piriform cortex 4 mixed cortex 5 hippo 6 thalamus 7 entorhinal cortex 8 cerebellum