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 -20C acetone.
  No permeabilization needed .
• 2.   Methanol Fixation Fix cells in -20C
  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
• 1200 or 1000 for 1oAb is often a good starting
  point
• 1200 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 cat715-165-150)
  or AlexaFlour 488 (goat anti mouse A11029 0.5 ml
  Invitrogen) diluted 1200 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 -gt 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, dont 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.