ALine Modular Device: Immunocytochemistry

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. If cells are only in the top well, there is no need to stain within the channel; however, the channel should still be washed.

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 serum for blocking

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 (store at 4ºC)                       for 25ml: 75 µl Tx-100 + 22.5 ml 1x PBS + 2.5 ml goat serum        (or other serum)

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

Method

  1. Wash cells with PBS (flush through bottom channel, pipet in and out of top well 3 times). Check channel for bubbles.
  2. Fix cells by adding 50 µl warm fixing solution or PFA to top well. If cells are in channel, add 10 µl to bottom channel. Check channel for bubbles. Incubate for 5 min in the incubator (37ºC, 5% CO2).
  3. Remove fixing solution (save as haz. waste).
  4. Wash well (100 µl) and channel (20 µl) with PBS. Check channel for bubbles.
  5. Permeabilize cells by adding 50 µl 0.1% Triton-x 100. If cells are in channel, add 20 µl to bottom channel. Check channel for bubbles. Incubate for 1 min at RT.
  6. Quickly wash well (100 µl) and channel (20 µl) with PBS. Check channel for bubbles.
  7. Block cells by adding 50 µl appropriate blocking solution in 1X PBS. If cells are in channel, add 20 µl to bottom channel. Check channel for bubbles. Incubate for 30 min at RT.
  8. Wash well (100 µl) and channel (20 µl) with PBS. Check channel for bubbles.
  9. Add 1º antibody(ies) and incubate at 4ºC overnight or 1-2 hr at RT. Perfuse antibody solution in both the top well (50 µl) and bottom channel (20 µl). Check channel for bubbles.
    1. Make 1º antibody on ice and store in fridge before use.
    2. For no antibody and 2º only controls, add 100 µl blocking solution.
  10. Remove 1º antibody.
  11. Wash well (100 µl) and channel (20 µl) 3x with PBS, 5 min each wash. Check channel for bubbles.
  12. Add 2º antibody(ies). Perfuse antibody solution in both the top well (50 µl) and bottom channel (20 µl). Check channel for bubbles. Incubate 1 hr at RT. Keep away from light.
    1. Make 2º antibody on ice and store in fridge covered from light before use.
    2. For no antibody controls, add 100 µl blocking solution.
  13. Remove 2º antibody.
  14. Wash well (100 µl) and channel (20 µl) 3x with PBS, 5 min each wash. Check channel for bubbles.
  15. Add 50 µl Hoechst 33342 to top well (or follow protocol for Dapi stain). If cells are in channel, add 20 µl to bottom channel. Check channel for bubbles. Incubate 3 min at RT. Keep away from light.
  16. Remove stain.
  17. Add PBS (100 µl top and 20 µl bottom) to prevent cells and membrane from drying.
  18. Image immediately or store at 4ºC until imaging.

Notes: 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 blocking solution and antibodies. If you get bubbles, remove by pipetting 100 µl more aggressively. Take extra precaution when adding fixing solution into channel.