Immunocytochemistry (ICC) Visual Protocol

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In the immunocytochemistry visual protocol video, you will learn how to coat coverships, prepare cell culture plats, plate cells for ICC, fix, permeabilize, block and add a primary antibody to your plated cells, add a secondary antibody, wash the plated cells, double label and mount your coverslips for analysis. Download PDF or request a print copy of our complete guide to multicolor ICC/IF by visiting www.novusbio.com/support/immu... Full length protocols and additional help can be found in the support section of www.novusbio.com, through our live chat service, or by calling us directly to talk with our elite customer and technical service scientists.

[@beardog6569]

I never knew that the different roles of Triton X and Tween were to target different regions within cells. Thank you!

[@osefelix]

thanks a lot for this

[@YablonskaOksanaSevama]

very nice! thanks!

[@songthanh896]

Thank you very much 😊

[@fatimadenouden9655]

How much antibody solution do you add per well? Since antibodies are expensive I want to add as less as possible, but I want to make sure it is enough.

[@joe30025]

Thanks for the great guidance, what is the difference if we grow the cells directly on the well plate and capture the ICC from there instead of using cover slip?

[@NovusBiologicals]

Here is the link for Novus' guide to ICC/IF which focus on the fundamentals of multicolor ICC/IF, including detailed protocols and troubleshooting tips: www.novusbio.com/support/immunocytochemistry-icc-handbook

[@ArshadPadhiar]

thank you so much,
Question: I want to target two diffrent epitopes at same time,one target present on cell surface, and the other in Golgi apparatus. so, if i fix my cells with parafromaldehyde, this will maintain the integrity of the cells but i have to pemeablize with some detergent, to see my intracellualar protein which results in the disruption of memb protein, and if i fix it with methanol or acetone, which later not require permeab, but it can precipitate the cell surface proteins.

[@Yhusi]

Thanks for the guide, how to avoid the cells grow in the bottom of the cover slip after 7 days of differentiation? Thanks in advance for your help

[@ArshadPadhiar]

so can i target two of the epitopes at same time, or i have to see them separately,?

[@marianur71]

Beautiful

[@ottorose832]

we like your green gloves Sir

[@user-rx1vo8rl9t]

can you tell me if there is risk of photobleaching with exposure of stained slides to room light and for how long?

[@InfinitiDreamerAkhshu]

can we coat the coverslips with collagen directly

[@zhoutui]

6:52 using a different fluorophore not a floor for it.

[@mohammedally2527]

Why do we grow the cells in a well containing a coverslip? does it act as a way to keep the cells in better condition ?

[@NovusBiologicals]

Cells are grown on coated coverslips (i.e. Poly Lysine - www.rndsystems.com/products/cultrex-poly-d-lysine_3439-200-01 or Fibronectin - www.rndsystems.com/products/recombinant-human-fibronectin-protein-cf_4305-fnb), often in a 24 well plate for ICC experiments. This is to allow for cell adhesion for your staining experiments. Due to the reduced number of cells required for ICC, plating cells on coverslips provides space for cells to become adhered and confluent, providing maximum sample conditions for your ICC staining. You would remove the coverslips after the cells that adhere to the coverslip are confluent. Growing cells on coverslips allows ease of staining and mounting onto a slide for your microscopy imaging after experimental conditions are provided. It also provides a way for you to be able to do variable experiments on cells (i.e. control vs treated) at the same time in different wells/coverslips. You can then mount both conditions on a single microscope slide, stain them at the same time, and get your imaging results at the same time as you know that all cells are treated to the same staining conditions. Different staining conditions, particularly antigen retrieval, can vastly impact your results and should be kept the same during staining groups you want to compare. For more information on ICC, browse our Protocols (www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-cells-coverslips), Troubleshooting Guide (www.novusbio.com/support/support-by-application/immunocytochemistry/troubleshooting.html) or ICC Handbook (www.novusbio.com/support/immunocytochemistry-icc-handbook).