First

steps in slide staining

Take slides out of the -80 freezer
o Let dry for 10-15 mins
o Put them in the slide chamber
o Add PBS, place on he shaker, and let wash for 15 mins
Make 50 mL of 0.2% triton
o 49mL of PBS + 1mL of 10% triton
o Place on shaker and let wash for 15 mins
Wash with PBS for 15 mins
o Suction PBS and use marker to draw circle around slides
o Keep some PBS on slides so tissue doesnt dry out
Make 5% Normal Donkey Serum (NDS)
o NDS found in -80 freezer
o 5mL of PBS w/250 microLiters of NDS
o Get rid of remaining NDS (sometimes there isnt any leftover)
o Let sit for 1 Hour in the petri dishes with the tissue water
Make Anti-body buffer
o 0.2g of BSA (found in giant container in the 4 deg freezer)
o add BSA to 20mL of PBS + 200 microLiters 10% triton
o Spin and shake for 30 mins
Use 4mL of anti-body buffer + 8 microLiters of Guinea pig anti-insulin
antibody + 8 microLiters of Anti-Ki67
o Leave over night