Griffin:Immunofluorescence Cell Staining

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Below is a simple and clear approach to immunofluorescence. There are 3 common fixation methods listed below that are proven and effective for a multitude of adherent and suspension cells. 1% formalin PBS is a good starting fixation method.For a typical slide, it takes ~300 microliters to cover an area equal to 2 coverslips.

I) Culture cells to 50-70% confluency. Some cells (endothelial/epithelial) may require a higher level of confluency. For suspension cells in a regular petri dish or flask, transfer enough cells suspended in medium to each well of a poly-D-lysine-coated microslide. Then Incubate microslide in CO2 incubator from 1 hour to overnight to allow cells to attach to the bottom of the slide. The incubation time varies depending on the suspension cell used. Check for cell attachment using a microscope.

Gently aspirate medium from the wells, wash the attached cells briefly with 1x PBS and proceed with the Cell Staining Procedure of choice (i.e., protocol 6: Immunoperoxidase Cell Staining, or protocol 7: Immunofluorescence Cell Staining)

II) Fix the subconfluent cells appropriately, followed by 2 PBS washes (dipping the slides gently into separate beakers of PBS is one way to wash).

III) Blocking: Gently pipet 10% BSA, PBS onto the slide and incubate for 30 minutes rt.

IV) Primary Antibody Incubation: 1:50 in 2% serum/PBS for 60 minutes at room temperature followed by 3 separate and gentle PBS washes.

V) Secondary Antibody Incubation: 1:100 in 2% serum/PBS for 60 minutes at room temperature followed by 3 PBS washes and 1 dH2O wash.

At this point prop the slides at an angle to allow them to partially dry. (You will see liquid evaporate slowly off slide when faced up. Wait for all liquid to evaporate and then add the mounting medium) approximately 5 ul drop (no air bubbles) to each area (8 areas for 8 well plates) Gently drop the coverslip onto the slide and align. Allow it to settle. Place mounted slide in light proof box and place in 4 degree overnight priot to performing the visualization.

The mounting agent is two component, one powder and one liquid. When you add the liquid make sure all the powder is dissolved. The powder tends to be in chunks on the bottom of the the jar vial. Vortexing may not loosen up this chunk and into its dissolved state. Also let the soultion set for 5 minutes before pipeting to allow air bubbles to rise out of the glycerol. Molecular Probes Prolong Antifade Kit

Visualize under the appropriate fluorochrome filter.

Common fixatives to consider in protocol optimization

(1) Methanol fixation (for cytoskeletal components): The methanol fixation is an easy method; however, it frequently solubilizes and removes membrane bound antigens. By a simple precipitation of the protein, methanol only provides low structural preservation.

  • Rinse the cover glass with PBS.
  • Fix cells by incubating the cells in pre-cooled 100% methanol at -20 oC for 10 min.
  • Wash cells with PBS.

(2) Formaldehyde fixation (for membrane associated components): Higher polymers, which are initially insoluble, are sold as a white powder known as paraformaldehyde.

  • Rinse cells with PBS at room temperature.
  • Fix in 3-4 % paraformaldehyde in PBS for 15 min at room temperature.
  • Wash 3-times 5 min each with PBS containing 100 mM glycine.
  • Permeabilize cells with 0.1% Triton X-100 in PBS for 1 to 4 min.
  • Rinsed with PBS.

(3) Formalin fixation (for membrane associated components): The liquid known as formalin contains 37-40% of formaldehyde and 60-63% of water (by weight), with most of the formaldehyde existing as low polymers.

  • Rinse cells with PBS at room temperature.
  • Fix in 1 % Formalin in PBS (prepared from 10% buffered formalin PBS) for 10 min at room temperature.
  • Wash 3-times with PBS
  • Permeabilize cells with 0.1% Triton X-100 in PBS for 1 to 4 min.
  • Rinsed with PBS.

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