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Staining Protocol 3D MPS Devices (Version 1.0) | ATLAS-D2K Center

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

Staining Protocol 3D MPS Devices (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 immunostaining cells seeded and cultured in a three-dimensional microvascular culture system.

Procedure

  1. Fix devices with 2% PFA/4% sucrose for 20 minutes by placing fixative into device inlet and outlet.
  2. Remove fixative from inlet and outlet.
  3. Wash 3x10 minutes with 0.5% PBST by placing into device inlet and outlet.
  4. Permeabilize cells with 0.1% Triton X-100 for 10 minutes at room temperature by placing Triton into device inlet and outlet.
  5. Wash 2x10 minutes with PBST (as above).
  6. Perform one long wash 30 minutes with PBST (as in Step 3).
  7. Apply Background buster (Accurate Chemical NB306) for 60-90 minutes at room temperature.
  8. Remove Background buster and wash 1x10 minutes with PBST .
  9. If necessary, perform appropriate Avidin/Biotin blocking (Vector SP-2001 ) and/or Rabbit Fab Blocking (Jackson Immunoresearch 111-007-003 & 011-000-007) for 30 minutes each, washing 1x10 minutes with PBST between each block.
  10. Add approximately 400ul of primary antibody at appropriate dilution (all antibodies diluted in PBS with 2% BSA) overnight at 4°C.
  11. Wash devices 5x10 minutes with PBST.
  12. Add 200ul of secondary antibody at appropriate dilution for 60 minutes at room temperature.
  13. Add 200ul of appropriate fluorescent detecting antibody for 60 minutes at room temperature.
  14. Wash devices 3x10 minutes with PBST.
  15. Add 200ul Phallodin (Fisher A22287)/DAPI (Sigma 1023676001) for 60 minutes at room temperature.
  16. Wash devices 3x10 minutes with PBST.
  17. Store at 4°C with PBS for up to one week (check daily to make certain devices are hydrated).
  18. Image devices on confocal microscope.

Notes:

  • Devices can be fixed for up to 1 week before staining.
  • Image devices as soon as possible following staining.
  • Imaging on confocal requires adjustments to microscope objectives.

Consortium

(Re)Building a Kidney (RBK) Consortium