实验概要

Immunofluorescence  is a technique used for light microscopy with a fluorescence microscope  and is used primarily on biological samples. This technique uses the  specificity of antibodies to their antigen to target fluorescent dyes to  specific biomolecule targets within a cell, and therefore allows  visualisation of the distribution of the target molecule through the  sample. Immunofluorescence is a widely used example of immunostaining  and is a specific example of immunohistochemistry that makes use of  fluorophores to visualise the location of the antibodies.

Immunofluorescence  can be used on tissue sections, cultured cell lines, or individual  cells, and may be used to analyse the distribution of proteins, glycans,  and small biological and non-biological molecules. Immunofluoresence  can be used in combination with other, non-antibody methods of  fluorescent staining, for example, use of DAPI to label DNA. Several  microscope designs can be used for analysis of immunofluorescence  samples; the simplest is the epifluorescence microscope, and the  confocal microscope is also widely used. Various super-resolution  microscope designs that are capable of much higher resolution can also  be used. 

主要试剂

384-well view plates (Aurora)

HUVEC (pooled, Lonza)

EBM-2 Medium (Lonza)

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

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

Donor Calf Serum (DCS)

Triton® X-100

Primary antibody (Sigma)

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

Hoechst 33342

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.