2. To perform in situ studies on mRNA or DNA, cells morphology and genetic information has to be preserved. Fixation(preservation) is conducted with 3.4 % formaldehyde – It forms a chemical bridges between proteins and cross-links mRNA and DNA with chemical groups of proteins You will follow a protocol as for a cell passing. 1) Dislodge cells from a surface with TRYPSIN-EDTA 2) Place cells on slides 3) Conduct fixation- TOMORROW 4) To maintain the line, we will freeze cells

3. Group 1Group 2Group 3Group 4 One person Big dish Group 1 and 2 (1 person) makes 4 slides (one for each group member) Cell Freezing

4. Clean all surfaces and labware/chemicals you will use under the hood. Everything that goes into the hood has to be sterilized with 70% ETOH. Take Trypsin EDTA 0.25% solution, electronic pipette, PBS, marker pen, forceps, medium 75cm2 dish, glass slides(group 1, 2) 4 Freezing tubes (3,4) FBS:DMSO(3,4)

5. Group 1&2. Take a big 75cm 2 dish and put 2 glass slides inside, with forceps. Add 23 ml of fresh medium. Shake to let medium cover the slides completely Aspirate used medium from old flask and wash cells twice with PBS. Add 1ml of Trypsin-EDTA. Wait a minute while tilting the flask a bit. Remove the trypsin. Close the flash and check under the microscope if cells are mobile (hit the flask) Add 10ml fresh medium, “go up un down” few times until cells are well mixed Take 3 ml of suspended cells and drop by drop, put them on the 4 glass slides. Cells will attract to the bottom of the slides and adhere to them Close the dish and write the group number, medium, cell type and date The cells will adhere over night (until tomorrow)

6. Group 3&4 – HOOD WITH BURNER ! Prepare a 10 ml of FBS:DMSO(10%) mixture DMSO will replace water inside cells. This will prevent making ice crystals inside cells during freezing Aspirate used medium from old flask and wash cells twice with PBS. Add 1ml of Trypsin-EDTA. Wait a minute while tilting the flask a bit. Remove the trypsin. Close the flash and check under the microscope if cells are mobile (hit the flask) Add 10ml of FBS:DMSO(10%), “go up un down” few times until cells are well mixed Put 1.5 ml of cells in two freezing tubes Mark tubes with LINE,Passage,Date Put tubes in a temporary container in -80 degr. NEXT DAY: transfer cells to -150

7. Tomorrow Group 1,2 – fixing Group 3,4- Transferring to -150 Group 1 and 2 1) Prepare 3.7% Formaldehyde in DEPC-PBS by taking 1.5ml of 37% FA and 13.5 ml of DEPC-PBS in 15ml falcon tube. Put it to the freezer to cool down ( to 1 deg. approx). 2) Have ICE COLD PBS and Ice in the box prepared 3) Aspirate the medium from the big dish and wash cells 2x with Ice cold PBS 4) Put slides into the transport box (5 slides/box) and pour cold FA inside the container. Keep it on ice for 20min. - FIXATION 5) Remove the FA to the FA waste box and wash slides 2x with DEPC- PBS 6) Conduct the ethanol scale to remove H2O from cells. Start by pouring 70% ETOH for 5', replace it with 85% for 5' and 99% for 5'. 7) Mark slides (group,Line,pass,Date) and store in -20 deg.