The microscope has a high power lamp source, usually a mercury or xenon arc lamp. An excitation filter transmits the band of the excitation radiation. The excitation radiation is reflected by the dichroic mirror towards the condenser/objective lens that focuses the light on the specimen. Light emitted by the fluorescent molecules (higher wavelength due to Stokes shift) is collected by the same lens and is transmitted by the dichroic mirror towards the ocular lens. Immunofluorescence, that makes use of the very high specificity of antibodies towards their targets, is a very useful method for studying cellular markers and organelles. Immunofluorescence microscopic analysis of cell surface markers is straightforward wherein the cells are treated with the fluorescently labeled antibodies and studied under microscope.
Materials and Instruments
1. Methanol
2. Acetone
3. PBS (1X)
4. 1% Triton X-100
5. BSA (Fat free, acetylated): Prepare 5% BSA solution in PBS and filter with the 0.45mm filter to rmove particulate matter.
6. Primary antibody (anti-protein): An antibody can be developed against protein (antigen of interest) in rabbit or mice.
7. Secondary antibody: An antibody coupled with fluorescent marker (such as FITC) and directed against mouse IgG.
8. Epi-fluorescence microscope
Procedure
1. Fixation: This is the first steps and it is required for two purpose. (1) Stopping biological actrivity and (2) it stops the relative movement of cellular components and intracellular macromolecules. In addition, it reduces the damage to the cellular system and morphology. Fix the biological sample with Methanol: Acetone (7:3) mixture at -200C for 15 min. Hydrate the sample with 1X PBS.
2. Permeabilization: Cell membrane is non-permeable to the charged as well as macromolecules. Only small molecule or hydrophobic dyes can pass through the membrane and reach to the inner compartments of the cell. Hence, cellular membrane needs to make porous by partically removing lipids from them. This process is known as permeabilization. Cells are permealized with 1% Triton X -100 for 15 min at room temperature.
3. Blocking: The intracellular spaces contains several antigenic sites and these need to block to reduce non-specific binding of the primary antibody. The cells are incubated with 5% BSA in 1X PBS for 15 min at room temperature. This step will allow masking of non-specific antigenic sites.
4. Primary Staining: Incubate the sample with primary antibody (1:50 in 2% BSA) for overnight at 40C or 1hrs at 370C. The primary staining at low temperature reduces the background signal and give good staining for sample where as staining at room temperature gives more amount of non-specific signal.