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Immunofluorescence Guide


Immunofluorescence (IF) is a common morphological approach used to determine the distribution of subcellular components. Antibodies that conjugated with fluorescent dyes are required in IF assay. The antibody specifically recognizes the antigen by binding to the epitope of target, and the fluorophore will be detected under a fluorescent microscope. Hence, subcellular components can be visualized in a dark background. IF can also be used as an alternative semiquantitative analysis method to monitoring the expression of the interest.

There are three types of IF: direct IF, indirect IF and combined IF.

Direct IF is using a single primary antibody that is conjugated with fluorescent dye.

Indirect IF is using two antibodies for the staining: primary antibody that specifically binds to epitope and a matched secondary antibody conjugated with fluorescence dye.

Combined IF is a combination of direct and indirect IF staining.

Table 1. Comparison of direct, indirect and combined IF.

IF type Direct Indirect Combined
Schematic diagram Immunofluorescence Guide Immunofluorescence Guide Immunofluorescence Guide
Advantages
  • Rapid staining
  • Available for antibodies from the same host
  • Less non-specific background signal
  • Secondary signaling amplification
  • A single secondary antibody can detect multiple primary antibodies from the same host
  • A primary antibody can be matched to diverse secondary antibodies with different fluorescence dyes
  • Secondary amplification for weak signaling
  • Available for antibodies from the same host
Disadvantages
  • Less sensitive because of lack of secondary signaling amplification
  • Time-consuming
  • Require antibodies from different hosts
  • Possibility of antibody cross-reactivity
  • Multiple-step staining

Among the three types of IF, indirect IF method is most popular.

IF approach can be used on tissue sections, cultured cell lines and individual cells. The process of IF is similar to Immunohistochemistry (IHC).

  • Sample collection and fixation

  • Samples must be fixed rapidly after tissue removal, and it is better to perform pre-fixation though heart infusion with 4% formaldehyde or paraformaldehyde in small animals like rodents. It is recommended that the tissues are no thicker than 10 mm and the volume of the fixative should be at least 15-20 times larger than the volume of the tissue. Fixation is very important for keeping the morphology and structure of cell as well as the integrity of antigen. Thus, fixation solutions must be carefully chosen according to different antigens and tissue samples.

Table 2. Fixation strategy for partial antigens.

Antigen Fixation solution Fixation condition
Most protein 95~100% alcohol
4% paraformaldehyde
3~10 min at 37℃
4~24 hr at 4℃
Enzyme Acetone 15 min at RT
Hormone 95% alcohol plus 1~5% glacial acetic acid 30 min at 4℃
Immune globulin 95% alcohol
Carbon tetrachloride
10 min at 37℃ then 15 min at 4℃
Fibrous protein 95% alcohol plus 1~5% glacial acetic acid 10 min at 37℃ then 15 min at 4℃
Virus Acetone
Carbon tetrachloride
Alcohol
5~10 min at RT then 30~60 min at 4℃
Polysaccharide and bacteria Acetone
10% formaldehyde
Methanol
3~10 min at RT then 30~60 min at 4℃
Lipoid 10% formaldehyde 3~10 min at RT
Cultured cell Warmed 4% paraformaldehyde 15~20 min at RT
  • Dehydration and embedding

  • Dehydration is required in preparing tissue sections for the following reasons:
    • Paraffin section: Paraffin is immiscible with water.
    • Frozen section: Frozen-thawed ice crystals would destroy the morphology of cells.

    Dehydration is always performed by immersing the tissue in a serious of increasing gradient ethanol solution or sucrose solution.
    Subsequently, tissue samples can be embedded by adding molten paraffin wax for paraffin sections, while OCT compound is added for frozen sections. This step provides proper hardness for soft tissue samples and allows the tissue to be cut easily.
  • Section and staining

  • Embedded tissues can be sectioned to thin slices with microtome or freezing microtome. The thickness of slices should be decided according to the cellular diameter and the purpose of IF assay. Thinner slices (≤10 μm) are suggested to directly mount to adhesive slides before staining, as they are easy to be staved in the multiple washing steps. Thicker slices (10~30 μm) will achieve better images by using free floating method, as the primary antibody could penetrate though both sides of the slice. And free floating sections are mounted to slides after staining. Free floating sections of small tissues such as mouse dorsal ganglia root (DRG) are difficult to perform and easy to lose sample. Thus, stick section method is recommended on some small tissue samples. The staining steps should be performed in dark when an antibody conjugated with fluorescent dye is involved.
    Reach out to IF protocols:
  • Imaging and analysis

  • Positive signaling is virtualized under a fluorescent microscope in a dark background. The location of interest is determined usually by co-staining of a protein of which the location has been known. Alternatively, the amount of positive cells or the fluorescence intensity of positive signaling could be measured for quantitative analysis. For instance, the stronger fluorescence intensity refers to a relative high expression of the target protein.

Workflow of IF on tissue sections

Figure 1. Workflow of IF on tissue sections.

IF and IHC both are powerful approaches for morphology analysis with important diagnostic and prognostic applications. Several differences must be concerned in your research:

Table 3. Comparison between IHC and IF

  IHC IF
Labeling Method Chromogenic Fluorescent
Processing step More as substrate required Less
Image
  • Ultra sensitive signaling
  • "fuzzy" reaction result from precipitate formed by chromogenic enzyme complex
  • High resolution
  • Multiple staining
Microscope Light microscope Fluorescent microscope
Stability Stable for years Less stable because of photobleaching
Example
(mouse hippocampus)
Immunofluorescence Guide Immunofluorescence Guide

References:

1. Fritschy, J.-M. and Härtig, W. 2001. Immunofluorescence. eLS. .
2. Grizzle, W.E., Special symposium: fixation and tissue processing models. Biotech Histochem, 2009. 84(5): p. 185-93.
3. Johnson, G.D., et al., Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy. J Immunol Methods, 1982. 55(2): p. 231-42.

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