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Flow Cytometry Protocol

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This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.

Preparation

  1. Harvest and wash the cells and determine the total cell number.
  2. Resuspend the cells to approximately 1×106 cells/mL in ice cold PBS. Add the appropriate volume of paraformaldehyde to obtain a final concentration of 4%.
  3. Fix for 10 minutes in a 37°C water bath.
  4. Wash cells twice with PBS containing 0.5% BSA.
    Note: For extracellular staining with antibodies that do not require permeabilization, proceed to step 8; for intracellular staining, proceed to step 5.

Permeabilization

  1. Add ice-cold 90% methanol (approximately 1mL per 1×106 cells) and vortex.
  2. Permeabilize for a minimum of 10 minutes on ice.

Immunostaining and Analysis

  1. Wash cells twice with PBS containing 0.5%BSA.
  2. Resuspend 1×106 cells in 100μL PBS containing 0.5%BSA.
  3. Add the primary antibody and incubate for 1 hour at room temperature (RT).
  4. Wash cells twice with PBS containing 0.5%BSA.
    Note: If using a fluorescent primary conjugated antibody, skip to step 14.
  5. Resuspend cells in fluorochrome conjugated secondary antibody diluted in PBS containing 0.5% BSA.
  6. Incubate for 30 minutes at RT.
  7. Wash cells twice with PBS containing 0.5% BSA.
  8. Resuspend cells in 0.5mL PBS and analyze on flow cytometer.

Immunofluorescence Protocol for Paraffin Embedded Tissue

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This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.

Tissue Preparation (Formalin Fixed, Paraffin-embedded Sections)

  1. Cut sections at 4μm and place on pre-cleaned and positively charged microscope slides.
  2. Heat in a tissue-drying over for 45 minutes at 60°C.

Deparaffinization and Rehydration

  1. Wash slides 2 times in Xylene for 3 minutes each time at room temperature (RT).
  2. Wash slides in Xylene 1:1 with 100% ethanol for 3 minutes at RT.
  3. Wash slides 2 times in 100% ethanol for 3 minutes each at RT.
  4. Wash slides 2 times in 95% ethanol for 3 minutes each at RT.
  5. Wash slides in 70% ethanol for 3 minutes at RT.
  6. Wash slides in 50% ethanol for 3 minutes at RT.
  7. Rinse slides gently with running distilled water for 5 minutes at RT.

Antigen Retrieval

  1. Boil slides in 0.01M sodium citrate buffer (pH6) at 100°C for 15-20 minutes. Remove the slides from heat and allow them to stand at RT in buffer for 20 minutes.
  2. Rinse twice with Tris-Buffered Saline + Tween (TBST) for 5 minutes at RT.

Immunostaining

  1. Block with 5% serum or BSA for 2 hours at RT.
  2. Drain blocking buffer from slide.
  3. Incubate slides with the diluted primary antibody overnight at 4°C with gentle agitation.
  4. Wash slides 2 times with TBST for 5 minutes at RT.
    Note: If using a primary conjugated antibody skip to step 18.
  5. Incubate slides with diluted conjugated secondary antibody for 2 hours at RT with gentle agitation.
  6. Wash slides 2 times with TBST for 5 minutes at RT.
  7. Mount coverslips.

Immunofluorescence Protocol for Cell Culture

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This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.

Sample Preparation

  1. Coat coverslips with polyethylineimine or poly-L-lysine for 1 hour at room temperature (RT).
  2. Rinse coverslips with sterile Hydrogen peroxide (3×5 minutes each).
  3. Allow coverslips to dry completely and sterilize them under UV light for at least 4 hours.
  4. Grow cells on glass coverslips or prepare cytospin or smear preparation.
  5. Rinse briefly in Tris-buffered saline (TBS).

Fixation

  1. Fix the cells in either 3-4% paraformaldehyde in TBS (pH7.4) or ice-cold acetone or methanol for 15 minutes at RT.
  2. Wash the samples twice with ice cold PBS.

Permeabilization

If the target protein is intracellular it is important to permeabilize the cells. Cells that have been fixed with acetone do not require permeabilization.

  1. Incubate the samples for 10 minutes in TBS containing 0.25% Triton X-100 (or 100uM digitonin or 0.5% saponin).
  2. Wash cells in TBS 3 times for 5 minutes each.

Blocking and Incubation

  1. Incubate cells with blocking buffer for 30 minutes to block unspecific binding of the antibody.
  2. Incubate the cells with the primary antibody overnight at 4°C (in the dark if using a fluorescent primary).
  3. Remove the solution and wash cells 3 times with TBS for 5 minutes each. Note: Skip to step 15 if using a primary conjugated antibody.
  4. Incubate the cells with the secondary antibody for 1 hour at RT in the dark.
  5. Remove the solution and wash 3 times with TBS for 5 minutes each in the dark.

Counterstaining and Mounting

  1. Incubate the cells with DAPI for 1 minute.
  2. Rinse with TBS.
  3. Mount coverslip with a drop of mounting medium.
  4. Store in the dark at -20°C or 4°C.