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µSiM Immunocytochemistry Protocol

You should be able to follow any ICC protocol within µSiM devices. This is an example protocol with recommended volumes and some notes for working in devices.

Reagents

16% Formaldehyde Solution (Thermo Scientific, Cat# 28908), 4% Paraformaldehyde (PFA), or other recommended fixing solution

Triton X-100

10% normal goat serum or proper blocking solution

Primary and Secondary Antibodies

Hoechst or Dapi stain

Solutions

Fixing Solution (4% formaldehyde)                 for 40ml: 10 ml 16% formaldehyde + 30 ml 1x PBS

Blocking Solution                                              for 25ml: 75 µl Tx-100 + 22.5 ml 1x PBS + 2.5 ml goat serum

Hoechst 33342 (10 mg/ml stock)                   for 10 ml: 1 µl Hoechst + 10 ml 1X PBS

Method

  1. Fix cells by adding 50 µl fixing solution to top well and 20 µl to the bottom channel. Check channel for bubbles, especially if cells are cultured in the channel. Incubate for 2-10 min.
    1. Methanol fixation can be as short as 20 sec
  2. Wash well (100 µl) and channel (20 µl) 3x with PBS, 5 min each wash. Check channel for bubbles.
  3. Permeabilize and block cells by adding appropriate blocking solution and incubating for 20-30 min at RT. You can either perfuse the antibody solution into both chambers or solely the chamber with cells. Check channel for bubbles.
    1. Well volume: 75 µl
    2. Channel volume: 20 µl
  4. Add 1º antibody(ies) and incubate at 4ºC overnight or 1-2 hr at RT. You can either perfuse the antibody solution into both chambers or solely the chamber with cells. Check channel for bubbles. Make 1º antibody on ice and store in fridge before use. For no antibody and 2º only controls, add blocking solution.
    1. Well volume: 50 µl
    2. Channel volume: 20 µl
  5. Wash well (100 µl) and channel (20 µl) 3x with PBS, 5 min each wash. Check channel for bubbles.
    1. If antibody was added only to the well, you can do just one wash of the channel.
  6. Add 2º antibody(ies) and incubate 1 hr at RT. Keep away from light. You can either perfuse the antibody solution into both chambers or solely the chamber with cells. Make 1º antibody on ice and store in fridge before use, protected from light. For no antibody and 2º only controls, add blocking solution. From here on out, keep devices protected from light as much as possible.
    1. Well volume: 50 µl
    2. Channel volume: 20 µl
  7. Wash well (100 µl) and channel (20 µl) 3x with PBS, 5 min each wash. Check channel for bubbles.
    1. If antibody was added only to the well, you can do just one wash of the channel.
  8. Add Hoechst 33342 and incubate 3 min at RT. Keep away from light. You can either perfuse the solution into both chambers or solely the chamber with cells. Check channel for bubbles.
    1. You can also stain the nuclei during the secondary stain to save time.
  9. Add PBS (100 µl top and 20 µl bottom) to prevent cells and membrane from drying.
  10. Image immediately or store at 4ºC until imaging. Store in a Petri dish wrapped in parafilm.

Notes:

As always, the channel volume is ~10 µl, but we recommend pipetting 20 µl to avoid adding bubbles into the channel.

Pipetting into the channel with PBS and solutions with detergent both have a different “feel” than pipetting media. PBS is less viscous and will not gather at the port. Detergent can be soapy. Due to these differences, it is more common to get bubbles during fixation and staining. If there are no cells in the channel, you can get away with adding PBS into the channel after fixing and then only staining in the well.

If you do need to stain in the bottom channel, pipet carefully through the bottom channel, holding the device at an angle when possible. Check for bubbles under the chip after each addition, especially when adding fixing and blocking solutions. If you get bubbles, remove by methods illustrated on this page (see “Bubble formation in channel“). In brief, either clear the channel and pipet back in 20-50 µl, or remove most liquid from the well and pipet 100 µl through the channel quickly, letting what runs out the port run into the well to avoid spills.

Take extra precaution when adding fixing solution into channel, as these solutions are often hazardous. It is helpful to use clamps, as illustrated on this page, to avoid having to directly handle the devices with gloves.