Department of Pathology, University of California, Irvine (UCI)
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Marchenko, S., Flanagan, L. Immunocytochemistry: Human Neural Stem Cells. J. Vis. Exp. (7), e267, doi:10.3791/267 (2007).
Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 μl of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.
Day 1: Preparing German Glass Coverslips and Seeding Cells
Note: German glass coverslips are often preferable for culturing primary neural stem cells and neurons. We use the following cleaning protocol to improve cell adhesion and spreading. Position coverslips in a small rack that will allow liquid access to both sides of the coverslip. First, incubate coverslips in 1% Liquinox for 10 minutes, followed by 3 washes with deionized water. Make sure that no soap bubbles remain. Next, incubate slips in 1M HCl for 30 minutes, followed by 3 washes with deionized water. Dry coverslips at 65°C overnight. Transfer coverslips to a glass dish and autoclave to sterilize.
Day 2: Fixing, Permeabilizing, Blocking, and Adding Primary Antibody to Cells
Note: We use the following 4% paraformaldehyde fixative to preserve cell morphology. The recipe includes a pH transition to allow the paraformaldehyde to go into solution. We prefer this method rather than the use of heat to dissolve paraformaldehyde because higher temperatures can lead to the generation of formic acid, which can increase background staining when using fluorescence. Some components of the fixative help to preserve cytoskeletal structure (MgCl2 and EGTA), while others help to maintain overall morphology (sucrose).
4% Paraformaldehyde Fixative for Cells
|Reagents and steps:||Amount for 100 mL of fixative:|
|MilliQ water||75 ml|
|Paraformaldehyde (weigh out in the fume hood)||4 g|
|10N NaOH, stir and add dropwise until solution clears||as needed|
|10X PBS||10 ml|
|1M MgCl2||0.5 ml|
|0.5 M EGTA||2 ml|
|6N HCl, titrate to pH 7.4||as needed|
|MilliQ water||bring to 100 ml|
|Store at 4°C for up to 1 week. Warm to 37°C before use|
Dilute the primary antibody (which is hopefully specific for the protein of interest) to a pre-determined concentration in 1% BSA/PBS (prepared by dilution from 5%BSA/PBS). Place diluted primary antibody (30 µl per 25 mm coverslip, 20 µl per 18 mm coverslip, 15 µl per 12 mm coverslip) on a glass plate covered with parafilm. Pick up the coverslip with fixed cells, invert such that the cells face down, and lay it over the antibody solution so that the cells are in contact with the antibody. Place a second parafilm strip over the entire construct. Incubate at 4°C overnight.
Day 3: Washing, Secondary Antibody Incubation, Nuclear Stain and Mounting
This protocol describes an immunostaining procedure for hNSPCs that minimizes the volume of antibody necessary and gives reliable cell staining. The procedure as described is best for intracellular antigens, but can be modified to stain cell surface molecules or to enhance staining of the cytoskeleton.
The authors would like to acknowledge Dr. Philip H. Schwartz of the National Human Neural Stem Cell Resource at the Children's Hospital, Orange County Research Institute for providing hNSPC cultures and Dr. Gary Bassell at Emory University for initial instruction in the parafilm staining technique.
|German glass (Deutsche Spiegelglas) coverslips||Tool||Carolina Biological||WW-63-3029||12,15,18, 22, 25 mm coverslips available|
|Liquinox phosphate-free detergent||Reagent||Sigma-Aldrich||Z273279||very viscous|
|Laminin, natural mouse||Reagent||Invitrogen||23017-015|
|EMEM (1X)||Reagent||Mediatech, Inc.||MT-10-010-CV||Distributed by Fisher under the indicated catalog #|
|24 well plate||Tool||Fisher Scientific||08-772-1|
|Triton X-100||Reagent||Fisher Scientific||BP151-500||very viscous|
|Paraformaldehyde||Reagent||Sigma-Aldrich||P6148||potential carcinogen, use caution when using it in powder and solution form|
|Bovine Serum Albumin, IgG-free||Reagent||Jackson ImmunoResearch||001-000-161|
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