实验概要

Immunofluorescence  allows the imaging of a specific factor in cells or tissue sections  through the use of a specific antibody chemically which is conjugated  with a fluorescent dye. Direct immunofluorescence staining has a primary  antibody labeled with a fluorescence dye whereas indirect  immunofluorescence staining has a secondary antibody labeled with a  fluorochrome used to recognize a primary antibody.

主要试剂

Phosphate Buffered Saline (PBS):

8.0 g NaCl

1.44 g Na2HPO4

0.24 g KH2PO4

0.2 g KCl

Add ddH2O up to 1 L, pH to 7.2 with HCl

1%BSA/PBS:

1 g of BSA in 100 ml PBS

实验步骤

1. Sample preparation:

1)         Grow cultured cells on chamber slides overnight, or add appropriate  amount of cells to poly-L-lysine coated chamber slides and incubate at  least 30 min at 37°C, at the time of fixation cells should be ~50%  confluent.

2)         Rinse cells briefly in PBS.

3)         Fix cells by incubation with 4% Paraformaldehyde, in PBS for 15 min at room temperature.

4)         Rinse three times in PBS, 3 min each.

5)         Add ice-cold acetone and incubate at -20°C for 10 min.

6)         Rinse three times in PBS, 3 min each.

2. Sample Blocking:

Block  samples in 5% normal serum from same species as secondary antibody in  1% BSA/0.2% Triton X-100/PBS for 1 h at room temperature, or overnight  at 4°C.

3. Sample staining:

1)         Dilute the primary antibody to the recommended concentration/dilution in 1% BSA/0.05% Triton X-100/PBS.

2)         Add 200 µl per well (8 wells) to the chamber slides and incubate 2 h at room temperature, or overnight at 4°C.

3)         Rinse three times in PBS, 3 min each.

            NOTE: If using primary antibodies directly conjugated with FITC or Alexa Fluor® 488, then skip to step 7.

4)         Prepare fluorochrome-conjugated secondary antibody antibodies in 1%  BSA/0.05% Triton X-100/PBS according to the recommended manufacturer  specification data sheet and add 200 µl per well (8 wells) to the  chamber slides.

5)         Incubate the samples for 1 h at room temperature in dark.

6)         Rinse three times in PBS, 3 min each.

7)         Coverslip with anti-fade mounting medium.

8)         Seal slides with nail polish.