Plant and Mammalian Tissue Culture

1. Plant and Mammalian Tissue Culture Immunolabeling Fixed Cells Experimental Design

2. Experimental Overview • Use fluorescent-labeled antibodies to immunolabel cells • Experimental Flow • Culture and fix cells • Permiabilize • Block non-specific binding sites w/BSA and serum • Primary antibody • Wash • Secondary Antibody • Wash • Mount and observe

3. Fixing Cultured Cells • Need to adhere cells to coverslip • Removal of calcium from solns will otherwise detach cells • Chemical Fixing involves covalent bonds between proteins on surface of cells and quartz or glass coverslip • Crosslinking agents (paraformaldehyde) forms amino groups on proteins to glass and each other • Causes alterations in potential antigens and may need to change based on need • Some methods work better with various cell lines

4. Fixing Cultured Cells 1.   Acetone Fixation Fix cells in -20°C acetone. No permeabilization needed . 2.   Methanol Fixation Fix cells in -20°C methanol. No permeabilization needed. 3.   Ethanol Fixation Fix cells in cooled 95% ethanol, 5% glacial acetic acid 4.   Methanol-Acetone Fixation Fix in cooled methanol. Remove excess methanol. Permeabilize with cooled acetone 5.   Formalin Fixation Fix cells in 10% neutral buffered formalin for 5-10 minutes. Rinse briefly with PBS. 6.   Paraformaldehyde-Triton Fixation Fix in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes 7.   Paraformaldehyde-Methanol Fixation Fix in 4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with cooled methanol for 5-10 minutes at –20 °C.   

5. Permiablization • Once cells are fixed, need to open cell to allow larger antibodies to enter cells • Triton X-100 – non-ionic detergent which forms pores in membranes and binds to hydrophobic lipids and proteins • or Digitonin – glycoside (large carbohydrate polymer) which precipitates cholesterol and solubilizes membranes • At higher concentrations, both surfactants will lyse cells!

6. Immunolabeling • Using antibodies to detect intracellular or cell surface antigens – can be protein, lipid, or other macromolecule targets • Blocking – use serum of secondary antibody to block non-specific binding sites • BSA – also binds proteins but poorly binds antibodies • Wash steps – include weak detergent and salt to remove poor binding proteins by defeating hydrophobic and small weak ionic (vanderwalls) interactions • Primary antibody (mouse, rat, human…) • Secondary antibody – must recognize primary Ab AND be conjugated (chemicaly covalently bonded) to a fluorophore (keep in mind the cube set!)

7. Immunolabeling • Dilutions – may vary best to trouble shoot the first time using • 1:200 or 1000 for 1oAb is often a good starting point • 1:200 for 2oAb

8. Experimental Plan • Label cells – two slips • One slip with both 1o and 2o Ab • Second slip without 1o • Difference will be due to specific binding of 2oAb to 1oAb • Which primary and labeled secondary are instructor dependent. Suggest using ERK from Santa Cruz Biotech for high signal.

9. Immunolabeling Protocol Cover slip - Use #1 thickness cover glass, either 12 mm round cover slips (cat 1943-10012 – Belco through fisher) or fisherbrand 22x22 (cat 12-542-B). - autoclave round cover slips in water in 100 ml beaker or in small beaker if slips are still on rack OR - autoclave square cover slips in Chex-all II sealing pouch (cat 024008). Keep flat. Culture – Culture cells as according to experimental needs. Seed at a medium density 50% (depending on cell type). Culture in 35 mm or 6 well plate. 1 square cover slip per well/dish 2 round cover slips per dish/well. One coverslip will have both primary and secondary antibody the other coverslip will not include primary antibody. Primary and Secondary Antibodies – staining can be performed with a variety of primary antibodies. For those looking for a strong signal in cytoplasm, ERK from Signal Transduction or Santa Cruz Biotechnology works very well. This is an abundant protein and the antibodies are very well. Another option would be b-actin or other antibodies that will label organelles.

10. Immunolabeling Protocol IMPORTANT POINTS Before starting and after fixing cells observe EACH cover slip. Look for intactness of cells. Focus on the shape and edges (margins) of the cells. Do they look dried out or hairy? If so than the cells are likely to have dried. If using round cover slips, identify the slip(s) to use for the rest of the labeling. Continue monitoring after digitonin/triton x-100. Cells, parafilm and media must all be at 37oC or the cells will round up! When rinsing, changing buffers or fixing cells, NEVER let the slip go dry. That is, only rinse or remove solution from 1 or 2 wells at a time. Even though moist, the cells will dehydrate and membranes become damaged. Carefully minimize the time the cells are out of a buffer/solution to seconds. “Incubation Chamber” For all incubations, cut a #1 Whatman filter paper to fit a 100 mm polystyrene dish, wet ½ of the filter paper with deionized water (the rest of the paper will become wet as the water wicks through the filter paper). Place on the bottom of the dish. Blot excess water from the plate with a kim wipe. Cover the moistened paper with a smaller sized square of parafilm. Mark the parafilm with a number to correspond with a cover slip. When incubating at 37oC, seal the chamber with parafilm. When doing this, hold the chamber firmly against your body to avoid sudden movements if (when) the parafilm rips. Pay attention to the orientation. Know what side of the slip has the cells. This is vital when washing the cells after primary Ab incubation.

11. Immunolabeling Protocol Processing Cells/Cover Slips (ALL media must be at 37oC to work. Keep cells in incubator as much as possible and do not place cells on bench or the solutions will quickly get cold and cells will lift). 1) Remove MOST not ALL of the media. Fix the cells on coverslips with ~1.0 ml of freshly prepared 3.7% paraformaldehyde in PBS for 20 minutes at 37oC. 2) Wash 2 x 2 min. with warmed Na2 buffer. 3) Permiabilize the cells with 0.1% Triton x-100 (~1 ml at 37oC) for 10 min. 4) Transfer the slips to the incubation chamber. Use a fine tip forceps to pickup slips (protect tip by covering with 200 ul pipet tip when the forceps are not in use). Wick excess moisture with a small wedge of #1 Whatman filter paper. Quickly add 3 drops of blocking solution (5% NGS/0.1% BSA/PBS) just about 100 ul. Incubate at RT for 30 min. Keep chamber covered when not manipulating slips.

12. Immunolabeling Protocol 5) Pick up each slip and remove blocking solution by wicking solution with filter paper. Only process one slip at a time. Use a Q-Tip to remove left over soln. from the parafilm. As soon as each slip is ready, incubate with the appropriate 1 Ab (50 l on the top of each coverslip) in 1% NGS/BSA/PBS at 37C for 1 hr. Incubate in the sealed chamber. 6) Rinse 3x, 10 min. each with PBS containing 0.1% BSA. For round coverslips, this is best done on a wide sheet of parafilm. Tape down parafilm flat on bench and number spots for coverslips. Create three rows of 400 ul of BSA/PBS per cover slip. Wick the soln from the coverslip using filter paper. Place (float) the coverslip on top, cell side down, onto the bubble of BSA/PBS. Keep the cover slip from sinking. If the slip insists on sinking, turn the slip upside right (cell side up) at the bottom of the bubble. When removing the cover slip, do so with a motion drawing back the cell such that the surface tension of the bubble pulls the liquid from the cover slip. Replace the slip directly onto the next wash bubble without wicking residual soln.

13. Immunolabeling Protocol 7) Remove the slip from the last BSA/PBS bubble and place cell side up in the appropriate spot in te incubation chamber. Incubate cells in 2 Ab for 1 hr at 37C. The 2 Ab is prepared in 0.1% BSA/PBS. Use CY3 conjugated donkey anti-mouse antibody (Jackson ImmunoResearch cat#715-165-150) or AlexaFlour 488 (goat anti mouse A11029 0.5 ml – Invitrogen) diluted 1:200 in 0.1% BSA in PBS. Re-moisten the filter paper at the bottom of the incubation chamber if needed. 8) Rinse each slip 3X, 10 min. each with PBS containing 0.1% BSA as described in step 6.

14. Immunolabeling Protocol 9) Mount the cover slip in a suitable mounting medium on slides. Use a drop the size of the circle shown to the right -> “o” . This is typically 5 to 8 ul. Place the slip cell side down, in a forward motion with the front edge of the slip dipping into the middle of the mounting medium. Wick excess (the liquid flowing out from under the cover slips and spilling one the sides of the cover slip) using a think pie shaped piece of filter paper. Let the water wick into the tip of the paper, don’t push the slip around. This will cause sheering of the cells. If using Prolong antifade, allow the solution to cure overnight. Do not use nail polish. If using VectaShield (Vector Labs) seal the edges of the coverslip at the end with nail polish. 10) Clean the coverslip after curing/drying the sealing mount. Do this by applying a moistened kimwipe. Use just the corner of a bent kimwipe. Do not rub, but instead, allow the weight of the kimwipe to apply the pressure. Simply remove salts in a circular motion with the tip of a bent or folded kimwipe.