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.
a.
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.
b.
Allow
sufficient time (> 5 m) for washing buffer to diffuse into the specimen
between each wash.
c.
Select
fluorophore with higher photostability against photobleaching, such as Alexa Fluor® series.
d.
Choose
fluorophores with distinct excitation and emission spectra to minimize signal
crossover if multiple fluorophores are used.
e.
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.
Do you have your own tips and tricks for staining
cells in AIM chips? Share them with the user community!
-Sei Hien-
Applications Development Scientist
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