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Embedding 3D MPS Devices for Sectioning (Version 1.0) | ATLAS-D2K Center

PLEASE NOTE: ATLAS-D2K closed July 31, 2025 and this website is for reference purposes only.

Embedding 3D MPS Devices for Sectioning (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Racheal Jappert; Jeffrey Pippin; Ying Zheng; Stuart Shankland

Keywords

[‘3D culture’, ‘immunocytochemisty’]

Subjects

[‘Cell culture’, ‘Immunological techniques’]

Release Date

2017-05-26

Abstract

Describes the method for embedding and cryosectioning a three-dimensional microvascular culture system.

Procedure

  1. Wash devices 3x 5 minutes with ~200ul room temperature PBS.
  2. Fix cells by applying ~200ul of 4% PFA (4% paraformaldehyde in PBS, Fisher NC9245948) + 5% Sucrose (Sigma-Aldrich S9378-1KG) warmed to 37°C for 20 minutes.
  3. Apply PBS to both inlet and outlet.
  4. Remove screws from device.
  5. Invert device.
  6. Place PBS on coverslip. Figure 1: Diagram of Steps 1 through 6
  7. Place your finger on coverslip and very gently push down, pushing the micropattern portion of device away from the bottom piece and lift the bottom piece off, keeping your finger on the coverslip the whole time. Figure 2: Diagram of Step 7
  8. Remove the excess collagen gel using a small spatula placed in between the cover glass and the plexiglass and pulling away from the cover glass around all sides of the cover glass.
  9. Slide the cover glass off, making sure you do not drag pieces of collagen over the gel containing the micropattern.
  10. Making sure there is no collagen gel on your spatula, use the tip to loosen the gel from the plexiglass, first using a tip-tap motion up and down, and then sliding the spatula smoothly around the entire edge of the coverslip. Figure 3: Diagram of Steps 8-10
  11. Gently remove gel, placing the flat/bottom side facing up onto a pool of PBS on the plate lid.
  12. Trim the gel using a razor blade, using the inlet and the outlet as guides (leave the inlet and outlet in place on those sides as guides). Then trim the other two sides. Figure 4: Diagram of Steps 11-12
  13. Place the trimmed gel, flat side facing up into a 6 or 12 well plate in a 1:1 mixture of OCT:PBS. Note the gel will float. Wrap plate with parafilm and place at 4°C overnight. Gel should sink.
  14. Transfer gel to 100% OCT and place at 4°C overnight.
  15. Transfer gel into Tissue-Tek Cassette and freeze in a 100% ethanol – dry ice water bath Figure 5: Diagram of Steps 13-15
  16. Network portion of gel is 100μm thick. Cut 8μm sections on cryostat.

Consortium

(Re)Building a Kidney (RBK) Consortium