Cell Staining Protocol

Fixed Cell Staining with EviFluor Quantum Dot Conjugates

This protocol describes fixation, permeabilization and labeling procedures for use on adherent cells cultured in 96 well plates or multiwell culture slides (refer to table 1 for recommended volumes). The protocol has been developed with PC-3 epithelial adenocarcinoma, NIH 3T3 fibroblast and MDCK epithelial cell lines for intracellular staining.

Required Materials

  • Fixatives:
    • Prepare 80 ml of 2% paraformaldehyde in PBS by diluting 10 ml of Paraformaldehyde 16% solution (Paraformaldehyde, 16% solution, EM Grade (Electron Microscopy Sciences, Inc., Catalog # 15700)) with 70 ml of PBS 1X.
  • Buffers
    • Prepare PBS by diluting 1 part of 10X PBS stock (10X Phosphate-buffered saline (PBS), pH 7.2. Sigma, Catalog # P5493) into 9 parts nanopure H2O.
  • Permeabilization buffer
    • 0.1% (v/v) Triton X-100 (Sigma Catalog # 93443, 10% solution) in PBS 1X.
  • Primary Antibody Blocking Buffer
    • Prepare 2% BSA, 1% Fish Skin Gelatin (Sigma # G-7765) and 0.1% Triton X-100 in 1X PBS.
  • EviFluor Blocking Buffer
  • Washing Buffer
    • 0.1% Triton X-100 in 1X PBS
  • Mounting media reagent
    • 50% glycerol in PBS.

Procedure

  1. Culture cells overnight to the desired confluence. See Table 1 for recommended volumes.
  2. Fixation
    1. Remove cell culture medium by aspiration and wash cells once with PBS.
    2. Add 2% formaldehyde in PBS for 10 minutes.
    3. Wash cells 3X with PBS ~3min each.
  3. Permeabilization
    1. Add 0.1% Triton-X in PBS and incubate for 15 min at room temperature (RT).
  4. Block
    1. Remove permeabilization buffer with pipette.
    2. Add Primary Blocking Buffer and incubate for 20 min. at RT.
  5. Primary Antibody Labeling
    1. Dilute primary antibody in blocking buffer. Concentration may need to be optimized for each antibody (refer to manufacturer's recommended dilution). A starting range of 1-10 µg antibody/ml has been used.
    2. Remove blocking buffer with pipette.
    3. Add primary antibody solution and incubate for 60 min. at 4°C on a rocking platform, or as recommended by the manufacturer.
    4. Remove primary antibody and wash cells 2x 3min. with Washing Buffer.
  6. Secondary Antibody Labeling with EviFluor
    1. Discard the Washing Buffer
    2. Block the cells with prepared EviFluor Blocking Buffer, incubate for 30 min. at RT.
    3. Dilute EviFluors in prepared EviFluor Blocking Buffer to a final OD of 0.01-0.005 at the peak absorption of the EviFluor (e.g. If the peak absorption of your EviFluor sample is 0.3 dilute 1:30-1:60). Pre-incubate EviFluors in EviFluor Blocking Buffer for 15 min. at RT, prior adding to the cells.
    4. Add diluted EviFluors to cells and incubate for 2 hours at 4°C on a rocking platform.
  7. Wash
    1. Wash cells 3 x 5min. with Washing Buffer.
  8. Prepare sample for imaging.

    • 96-well plate
      1. Add 30 µl of PBS, cover.
      2. Image within 12 hours for optimal results. Can be imaged after longer periods of time when stored at 4°C.
    • Culture Slide
      1. Add 50% glycerol in PBS (see table 1 for volume)
      2. Place coverslip over cells.
      3. After coverslip has settled, remove excess glycerol and seal edges with clear nail polish.

Table 1 - Recommended volumes per well for each reagent

Reagents (Volumes in µl) 96 Well Plate 8 Well Culture Slide 4 Well Culture Slide
PBS (Wash) 100 100 200
Fixative 100 100 200
Permeabilization Buffer 100 100 200
Primary antibody blocking buffer 50 100 200
Primary antibody in primary antibody blocking buffer 50 75 150
EviFluor blocking buffer 50 75 150
EviFluor in EviFluor blocking buffer 50 75 150
50% glycerol - 8-10 10-15

Order EviFluors Below

Product Availability
Conjugate Wavelength Part Number Concentration Price
Goat anti-Mouse 520nm
600nm
620nm
680nm
 EF-C11-GMO-0XXX 2.5uM $289
Goat anti-Rabbit EF-C11-GRA-0XXX $289
Goat anti-Rat EF-C11-GRT-0XXX $289
Streptavidin EF-C11-STR-0XXX $439
Biotin EF-C11-BT1-0XXX $439
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