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If staining for a phospho-protein, use TBST instead of PBST

1) Place sterile coverslips into each well of a 6well plate, with sterile forceps (use EtOH + Bunsen burner to sterilize forceps if no
autoclaved ones,  

      wait  for EtOH to evaporate before using the bunsen burner)
2) Seed 0.100-0.300E6 cells/well in a 6well plate on a sterile coverslip, let them adhere overnight or if treating with drugs treat for designated amount

      of time before the procedure.

3) Remove medium and RINSE QUICKLY but GENTLY 2 times in 1XPBS to remove media
      a. This step must be done quickly as cells WILL lift off and start dying, also be gentle when adding 1XPBS, if doing many plates at once, do it  

            sequentially, not ALL at the same time.
      b. PS: Have 4% Paraformaldehyde (PFA) Ready to go. (A good PFA to use is sold by Electron Microscopy Sciences Cat#15710)
4) Fix w/ 4% PFA (add approx. 1-2mL per well to cover the coverslip)
      a. Dilute down from 16% PFA vial w/ 1XPBS (so that 1XPBS in final volume)
        16% PFA   10mL
        10X  PBS    4mL
        1XPBS        26mL
5) Newtate for 10-20min @ RT
6) Wash 3x5mins with 1X PBS (discard is a designated PFA waste bottle!! Because PFA is nasty)

     a. At this point you can leave cells for 1-3 days @ 4deg (1 week if desperate but expect crappy results.)

7) If staining for a nuclear protein (if not skip this step):
      a. Permeabilize nucleus w/ 0.5% Triton X-100 in 1XPBS.
      b. Make about 50mL (2.5mL Triton X-100 in 50mL 1XPBS) and add 2mL/well
      c. Newtate 10min @ RT
8) Wash 3x5min with 1XPBS
9) Block for 1h @ 37deg w/ 5% BSA in TBST/PBST (~2ml/well)
10) Wash 3x5min with 1XPBS
11) Add 100uL of primary antibody in 1%BSA TBST/PBST to Waxy paper and FLIP slides FACE DOWN
      a. Leave overnight @ 4deg
      b. You should have a black container with a lid, with moist paper towel below the waxy paper on which your coverslips are resting to keep the
           whole thing moist

12) Get a new 6 well plate and label it accordingly, and flip BACK the slides into the plate.
13) Wash 3x5min with 1XPBS
14) Add 100uL of 2o Ab in 1%BSA TBS/PBS to waxy paper and FLIP again.
      a. [2o Ab] = 1:100
      b. Newtate for 1h @ RT
      c. Secondary antibody is attached to a flourophore! Everything must be done in a darkened env from this point on to avoid photobleaching.
15) Wash with 1X PBS for 3x5min
16) Apply Vectashield w/ DAPI onto slides. Do only 2-3 slides per time as Vectashield mounting medium dries VERY FAST.
      a. Take DAPI out of fridge at front of the lab (black box)
      b. Go to semi-dark environment (bring DAPI, Kimwipes, tweezers, slides)
      c. Use tweezers to carefully remove cover slip from well
      d. Put 1 drop of DAPI onto slide then put cover slip ontop, using tweezers to push down on slip to remove any bubbles.
      e. Seal with cheap nail polish sealer
17) Visualize with a confocal or another microscope.

Click Here to download the protocol

By: Pawel Buczkowicz

Modified by: Mark Barszczyk and Yev Chornenkyy

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