Bitan:parafin embedded tissue cutting and mounting

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Cutting (in ImagingCore) 1) gather materials (some which are in top desk drawer to the immediate left of the left microtome): 2 paper towels, 2 razors, a few paintbrushes of different sizes, bilious paper, roller, a few empty slides (usually on counter) 2) cut parafilm block from plastic encasement and peel off the plastic 3) draw a rectangle around the tissue -make sure to leave enough space around brain -make sure brain is positioned in bridge from -cut off a little notch at top left corner 4) put block into machine -make sure face is parallel with blade 5) unlock handle, turn on machine, press “HOME” 6) trim in larger sections until first reach the block 7) section at 8 um into ribbons -make sure the center of the block is cut first -use brush to brush waste into paper towel waste holder -cut slowly to prevent curtaining -to automate, change cut mode to Continuous, push the two “run” buttons at the same time -use brushes to move the ribbon (don’t pull too hard!) 8) if there are scoremarks on the tissue—stop! -brush off the face -wipe blade with kimwipe in one smooth movement upwards -move blade over if wiping does not work 9) after finish 1 board, check sections to see if hippocampus is all covered -turn on slide warmer -put a drop of CaCl in H2O on warmed slide and put section on top -check under microscope -can also put bilious paper on top of the slide and roll over it to get better view 10) note down the sections that don’t have any hippocampus (in beginning & end) and don’t mount those

Mounting 1) gather materials (Aida’s bench, top drawer with the red tape): thermometer, agarose, 2 razors, 2 paintbrushes, forceps 2) milliQ water in water bath (3/4th full), heat up to 45 degrees C (takes about 20 mins) 3) put in some agarose into the water and stir with paintbrush -make sure to stir/dissipate the bubbles in the water 4) label slides—write genotype (3Xtg, 2Xtg, or WT), mouse number, and slide number -make sure slide number is to the side so that they can be read in the slide boxes 5) cut brain section ribbons so that each slide will have 3-4 sections 6) mount slides -make sure the serrated edge is to the left -use forceps to hold sections (hold it by the very edge) -make sure no bubbles under sections (put in sections into water slowly) -easier if the forceps don’t get wet -use a paintbrush to guide section onto slide (hold it by the very edge) 7) slides will be dry in 1-2 hours (but make sure!) and can be put onto plate warmer (ImagingCore) for overnight

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