实验概要

We provide a protocol for fixation, immunostaining, and imaging in 384-well Plates.

主要试剂

Reagents

1. 384-well view plates (Aurora)

2. HUVEC (pooled, Lonza)

3. EBM-2 Medium (Lonza)

4. Fixation Solution - 16% Paraformaldehyde (PFA) aqueous solution (diluted to 4% in wells)

5. 1x PBS w/o Ca² and Mg² (prepared from powder)

6. Donor Calf Serum (DCS)

7. Triton® X-100

8. Primary antibody (Sigma)

9. Secondary Antibody (Alexafluor 647 goat anti-rabbit IgG–highly cross-adsorbed)

10. Hoechst 33342

Preparation Instructions

1. Triton X-100 Stock Solution - 10% (v/v) solution in PBS

2. Antibody Diluent - 10% DCS in PBS

3. Hoechst 33342 Stock Solution - 10 mg/ml in PBS

4. Permeabilization and Blocking Solution - 0.2% Triton X-100 and 10% Donor Calf Serum in PBS

5. Primary Antibody Solutions – Prepare 8 different primary antibody  dilutions ranging from 1:50 to 1:6,400 using Antibody Diluent.

6. Secondary Antibody Solution – dilute secondary antibody 1:1,000  using Antibody Diluent. Add Hoechst 33342 Stock Solution to a final  concentration of 1 mg/ml (10,000-fold dilution of Stock Solution).

实验步骤

1. Cells  preparation - HUVEC cells were cultured in EBM-2 medium and plated in  384-well plates at a density of 3,500 cells per well. The plates were  then incubated for 24–48 hours until 80–90% confluent before fixation.

2. Fixation

   1) Dilute Fixation Solution (16% PFA) 4-fold in test wells (17 ml of Fixation Solution added to 50 ml of medium in wells).
Note: Alternatively, Fixation Solution can be diluted to 4% PFA with PBS and added directly to wells after aspirating medium.

   2) Incubate for 20 minutes at room temperature.

   3) Wash once with 1x PBS.

   4) Store plates at 4 °C until ready to stain.

3. Immunostaining

   1) Wash once with 1x PBS and aspirate.

   2)  Add 50 ml of Permeabilization and Blocking Solution to each well.

   3) Incubate for 20 minutes at room temperature.

   4)  Aspirate Permeabilization and Blocking Solution and add 25 ml of Primary Antibody Solution.

   5) Incubate for 20 minutes at room temperature.
Note: In some cases, the primary antibody incubation time can be extended to increase signal intensity.

   6)  Aspirate Primary Antibody Solution, wash once in 1x PBS, and aspirate.

   7)  Add 25 ml of Secondary Antibody Solution.

   8) Incubate for 20 minutes at room temperature.

   9) Wash 4 times with 1x PBS.

   10) Store plates in 50 ml of 1x PBS at 4 °C until ready to image.

注意事项

Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.