0825-2016 Microdissection direct

12:00 get three slides of one case and two Rnase free PCR tubes out of refrigerator to room temperature, label tubes with 3G for glomeruli and 4T for tubules

finish lunch

13:45 go to microscope room with slides, PCR tubes, lysis buffer, pipette, tips, gloves, kim wipe, gabage bag and basket

14:00 start work

• turn on PC and lauch software
• turn on laser: anterior, posterior, key
• unload speciments (pay attention to the direction of slides)
• unload collectors: PCR tubes with 20ul lysis buffer in the bottom of caps
• modulate the codition (under no cap)
  camera/set up windows/draw in empty area/select white balance
• collectors: option/setting/learn by intelligent then the same to modulate next cap
• draw overview (prefer not to do this for saving time)
• under no cap (prefer with cap for good identification of the structure)
  • laser setting: power 40/aperture 3/speed 6/pulse frequency 1000 (better testing on intersticial area before using)
  • draw+cut glomeruli into 3G cap in one section
  • draw+cut tubules into 3T cap in one section
  • repeat the sections in one slides
  • repeat in all slides to collect as many targets as possible

unload PCR tubes (be careful, not drop out the tissues)

unload slides

exit software

turn off laser: key/posterior/anterior

shut down PC

switch off electricity

cover the microscope

cleaning IF necessary

sign out the utilising

check again

take all of my own materials and lock the room

when back to lab

• short spin down the lysis buffer
• add 80ul lysis buffer to each tube and spin again
• store in -80 centigrade refrigerator
• keep lysis buffer in cold room and arrange everything well