AIM chips are compatible with various imaging techniques, including fluorescence microscopy. In order to visualize the cells with fluorescence microscopy, you can use fluorescence-labelled cells (such as GFP-tagged cells) or stain the cells with fluorophore. Here are some tips and tricks for staining cells in AIM chips.


  1. Practice good fluorescence staining techniques to improve staining efficiency and minimize background.
    • Block the specimen with normal serum (or bovine serum albumin) from the same species as the secondary antibody is made from to reduce unspecific binding of the secondary antibody.
    • Allow sufficient time (> 5 m) for washing buffer to diffuse into the specimen between each wash.
    • Select fluorophore with higher photostability against photobleaching, such as Alexa Fluor® series.
    • Choose fluorophores with distinct excitation and emission spectra to minimize signal crossover if multiple fluorophores are used.
    • Use brighter fluorophores for less abundant antigens and vice versa.
  2. Do not use acetic acid in the fixation step if collagen gel I is used to fill the hydrogel channel. Acetic acid will dissolve polymerized collagen gel I.
  3. Account for the autofluorescence at the posts when selecting your fluorophores. The autofluorescence at the posts has a maximum absorption in ultraviolet range and emits blue fluorescence. The auto-fluorescescence at the posts can be used as reference points for the gel interface.
  4. Certain primary antibodies and dyes such as Phalloidin work best when they are incubated with the specimen at 4 °C for extended durations (>12 h).
  5. Set up differential volumes of staining solution between media channels if target cells are embedded in the 3D hydrogel. The interstitial flow created by the differential volumes will help to carry antibodies or dyes to the target cells.