實驗概要
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.
主要試劑
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
實驗步驟
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.
