Sample Preparation for ICC IF Experiments

Related Links

Fluorochromes Selection for Multicolor ICC/IF

Sample Preparation for ICC/IF Experiments

Fixation & Permeabilization

Blocking for ICC-IF Assay

Antibody Selection in ICC/IF

Detection Methods for ICC IF

Controls for ICC/IF Experiments

Counterstaining & Mounting

Useful Links

Multicolor ICC/IF Protocol

Troubleshooting ICC/IF

Secondary Antibodies

Organelle Markers Guide

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ICC Handbook

What do Consider Before You Prepare your Samples

Sample preparation is a critical step for achieving accurate expression and localization data in multicolor ICC/IF. Issues with sample preparation, fixation, and permeabilization can result in false or misleading staining. Before starting an experiment, review the scientific literature for information about the expression and localization of your protein of interest. If the target protein is not expressed under basal conditions, you may need to apply a stimulus (e.g. protein or chemical) to your cells to induce protein expression.

Another critical factor to consider is cell confluence. Typically, a cell density of 60-80% is optimal for ICC/IF assays. A high cell density can negatively impact cell architecture, whereas a low cell density may require imaging at a lower magnification or capturing multiple images to ensure a sufficient number of cells are imaged. Optimal cell density is also critical to maintain cell-to-cell contacts, and is of particular importance when studying proteins, such as cell junction markers, including claudin-1 or ZO-1.

Cells on Cover Slips or Slides

Adherent cells

Adherent cell lines are cultured on cover slips in multi-well plates using aseptic conditions. Coverslips must be sterilized using an autoclave or UV enabled laminar flow culture hood. Coverslips are then washed with ethanol in the culture hood and once dried, placed in culture wells using tweezers. If cells do not properly adhere to the surface, apply a coating matrix to the glass coverslips. The type of coating matrix used is dependent on the cell type, but poly-L-lysine works well for most cell lines. Other available matrix proteins are listed below*. Multiwell plate with coverslip having cultured cells

Non-adherent cells

Non-adherent cells are fixed after adding the fixative directly into the culture media at a ratio of 1mL of 4% PFA for every 1 x 106 cells. After 20 minutes of fixation, the fixative is removed and the sample is washed 2x with diH20. The fixed cells are suspended in diH20 and smeared on a gelatin coated slide using a clean micropipette tip. Smear preparation on a glass slide

*Cell type specific coating materials

Cell Types Suggested Coating Material
Embryonic stem cells Laminin
Primary keratinocytes/hepatocytes Collagen
Microvascular endothelial cells Gelatin
Neural stem cells Fibronectin