Immunocytochemistry: Human Neural Stem Cells

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Summary

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

Cite this Article

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Marchenko, S., Flanagan, L. Immunocytochemistry: Human Neural Stem Cells. J. Vis. Exp. (7), e267, doi:10.3791/267 (2007).

Abstract

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.

Protocol

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.

  1. In a tissue culture hood, place sterile coverslips in an appropriately-sized well plate.

  2. Coat coverslips with laminin for cell adhesion. Incubate coverslips in 10 µg/ml poly D-Lysine (PDL) in sterile water for 5 minutes at room temperature. Remove PDL and incubate coverslips in 20 µg/ml laminin in EMEM for 4 hours, or overnight, in a 37°C tissue culture incubator.


    1. Note: Refer to the Passaging Human Neural Stem Cells article (http://www.jove.com/index/Details.stp?ID=263) and the Counting Human Neural Stem Cells article (http://www.jove.com/index/Details.stp?ID=262) to learn how to resuspend and count hNSPCs.

  3. Rinse laminin-coated coverslips with PBS and seed cells into each coverslip-containing well at a certain density (we often use an initial plating density of 100,000-300,000 cells/ml for cells that we are plating for immunostaining).

  4. Place plate in 37°C tissue culture incubator and allow cells to adhere to coverslips (usually a minimum of 4 hours is required).

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
Sucrose 4 g
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
  1. On the bench, remove the media and add prewarmed fixative. Incubate for 5-15 minutes (depending on the antigen to be detected, we usually use 10 minutes). Perform this and the following steps at room temperature.

  2. Discard the fixative into an appropriate container, as many institutions handle paraformaldehyde as hazardous waste. Wash cells 3 times with PBS, 5 minutes each time. When removing solutions from the cells, be careful that suction does not dry out the cells. Also, always have the next solution on hand to add to cells before removing the solution that coats them so cells are not left to dry.

  3. If the antigen of interest is inside the cell, the cell membrane must be made permeable to allow entry of the antibody. To permeabilize cells, use a fresh 0.3 % Triton X-100 solution in PBS. Pipette slowly because Triton is viscous, and make sure the detergent is completely dissolved in PBS before adding to cells. Permeabilize cells for 5 minutes, followed by washing the cells 3 times with PBS, 5 minutes each time.

  4. The cells must be treated with a blocking agent to prevent non-specific binding of the antibody. We use bovine serum albumin (BSA) as a blocking agent. The blocking solution is made by dissolving 5% BSA into PBS (on a rocker or rotator because it takes time to dissolve) then filtering the solution with a 0.45 µm syringe filter. Block cells in 5% BSA/PBS solution for 1 hour at room temperature.

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

  1. Carefully remove the top parafilm strip and pick up the coverslip, invert it, and place it back into the plate with PBS in each well for washes so that the cells are facing up.

  2. Wash cells 3 times with PBS, 5 minutes each time.

  3. Choose a secondary antibody that will detect the primary antibody, for example, if the primary antibody was made in a rabbit, the secondary antibody should recognize rabbit IgG. Take care to also match the antibody isotype (IgG, IgM, etc). If using multiple primary antibodies, make sure they are from different species or isotypes, and plan to detect with different markers attached to the secondary antibodies. For example, you could use an anti-rabbit secondary coupled to a red fluorophore with an anti-mouse secondary coupled to a green fluorophore. Incubate cells in the secondary antibody solution diluted to an appropriate concentration in 1% BSA for 2 hours in the dark at room temperature, using the same technique as for primary (steps not shown in the video). The volume of secondary antibody needed for the incubation may be slightly larger than that for the primary antibody if the secondary is stored as a glycerol solution (40 µl per 25 mm coverslip, 30 µl per 18 mm coverslip, 25 µl per 12 mm coverslip).

    • Note: If you are using fluorescent molecules to visualize the secondary antibody, the sample needs to be protected from light once the secondary antibody has been added. Incubate in the dark, or in a foil covered plate.

  4. Carefully remove the coverslips from the parafilm as described for the primary antibody. Wash cells 2 times with PBS, 5 minutes each time, followed by a 1 minute incubation in 2 µg/ml Hoechst nuclear stain diluted in PBS (if a nuclear counterstain is preferred). If the Hoechst stain is old and the signal is too dim, you can increase the incubation time and/or the concentration. After incubation with Hoechst, wash 1 time with PBS for 5 minutes.

  5. The coverslips should be mounted on a slide with mounting media for visualization on a microscope. In cases in which a fluorescent molecule is used for visualization of the secondary antibody, the mounting media should contain agents to minimize photobleaching. We use Vectashield as a mounting media for fluorescence. Place an appropriate size drop of Vectashield on a microscope slide (12 µl per 25 mm coverslip, 6 µl per 18 mm coverslip, 3 µl per 12 mm coverslip).

  6. Carefully pick up the coverslip and rinse the back with deionized water to remove salts (from the PBS). Dab off excess water on a kimwipe or paper towel, and lay on the drop of Vectashield with cells facing down. Place the coverslip on at an angle and allow to descend slowly to avoid trapping air bubbles. Label the slide with the date and any sample information.

  7. Allow slides with coverslips to dry (in the dark) then suction off excess Vectashield from around the coverslip edge. The coverslip edges can now be sealed with nail polish, if desired (especially useful if coverslips will be viewed on an inverted microscope). We usually store our slides in a -20°C freezer.

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Discussion

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.

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Acknowledgments

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.

Materials

Name Type Company Catalog Number Comments
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
Poly-D-lysine hydrobromide Reagent Sigma-Aldrich P7280
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
H–chst 33342 Reagent Invitrogen H-1399
Vectashield Reagent Vector Laboratories H-1000 viscous

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References

  1. Flanagan, L. A., Chou, J., Falet, H., Neujahr, R., Hartwig, J. H., Stossel, T. P., Filamin, A. Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells. J. Cell Biol. 155, 511-518 (2001).
  2. Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. Regulation of human neural precursor cells by laminin and integrins. J. Neurosci. Res. 83, 845-856 (2006).
  3. Flanagan, L. A., Ziaeian, B., Palmer, T., Schwartz, P. H. Immunocytochemical analysis of stem cells. In Human Stem Cell Manual: A Laboratory Guide. Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. Elsevier/Cell Press. (2007).
  4. Marchenko, S., Flanagan, L. A. Passaging Human Neural Stem Cells. Journal of Visualized Experiments. 7, (2007).
  5. Marchenko, S., Flanagan, L. A. Counting Human Neural Stem Cells. Journal of Visualized Experiments. 7, (2007).

Comments

9 Comments

  1. I cannot view any video

    Reply
    Posted by: Anonymous
    October 11, 2007 - 6:24 AM
  2. Pretty good, but the network is too slow!

    Reply
    Posted by: Anonymous
    January 11, 2008 - 3:34 AM
  3. Sorry about that. What connection are you on?

    Reply
    Posted by: Anonymous
    January 11, 2008 - 7:51 AM
  4. Hello, I am a student in China, my major is pharmacology.Currently, I am buried in neuron cell culture and have to use immunocytochemistry method to mark and identify them.I am grateful for your video.I have learnt a lot  from your web . Now , I have a small question: if I use FITC dyeing, which step should I avoid light?just "add the secondary antibody"? if I have to avoid light, how? should I just use the silver paper around the coverslips? or should I doing the step in a dark room? Sincerely thank you for your work and hope for your feedback! Jing Wang

    Reply
    Posted by: Anonymous
    June 13, 2008 - 9:18 PM
  5. Hi Jing, You should avoid exposing your sample to light during secondary antibody incubation and while handling and storing your sample afterward.  It can be done by placing your sample into a lab bench drawer or wrapping foil around the sample.  For long term storage, we mount stained coverslips onto a microscope slide with an anti-fade Vectashield and store slides in a -²0C freezer Hope this helps, Good luck with your staining!

    Reply
    Posted by: Anonymous
    June 23, 2008 - 5:04 PM
  6. Hi, I am Jing. I have seen your satisfying answer and thankyou for your help!   good luck with your work!   Jing  

    Reply
    Posted by: Anonymous
    August 25, 2008 - 9:09 AM
  7. Hi, I am Grad student at Ball state and I am thinking to do the immunocytochemistry of GLC4 cells. I watched your video and it was mindblowing. Thank you very much for posting this video. I have a few query which are listed below: 1 Are the neural stem cells that you are working with a suspension cell? What modification would be there in the inital step if it is for GLC4 (a suspension cell)? ² You coated your cover slip with Poly D lysine later, isn't it? I assume it was not pre-coated from the manufacturer. Actually,  I was thinking to coat my slides with poly L lysine. Will it bring a good result just like in your case if I go for poly L lysine? Hope to hear and thanks again for all the information that  I could receive from you. Regards Kamal

    Reply
    Posted by: kamal p.
    October 22, 2008 - 7:31 PM
  8. This was a wonderful video and I thought the technique to reduce primary antibody usage was quite clever.  I have a technical question about the protocol: if I am using an antibody with a fluorophore (FITC) attached, will I still have good results if I rinse and mount the coverslip after the antibody is applied to the cover for ²4 hours?  Or is the technique from the video more successful for the longer, primary + secondary antibody protocols?  Thank you.

    Reply
    Posted by: Anonymous
    December 5, 2008 - 6:35 PM
  9. I work with hippocampal cultures. I follow similar steps for IHC as above..........however I use a higher percentage of Triton-X as my antigen is localized in the nucleus. Due to this my cells look rather deformed. Is the use of any other permeabilization agent suggested.

    Reply
    Posted by: Anonymous
    August 10, 2010 - 12:55 PM

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