What Is An Immunofluorescence Test Used For?

Introduction

Immunofluorescence (IF) is a powerful technique based on immunology, biochemistry and microscopy.

Its principle is to use fluorescently labeled antibodies as probes to locate and qualitatively analyze specific antigens in tissues or cells. IF is a good detection technology tool for studying the expression, location, distribution and migration of products of interest such as molecules, proteins or glycoproteins.

Experimental principle

The main principle of cell immunofluorescence staining is to use the specific binding between antigens and antibodies to display the target protein, mainly including the binding of proteins and primary antibodies, followed by the recognition and binding of secondary antibodies with fluorescent groups to the primary antibodies, and fluorescence can be observed under a fluorescence microscope.

Fixing cells with a fixative greatly increases the permeability of the cell membrane. When detecting cytoplasmic or nuclear proteins, a permeabilizer can also be used to further enhance the permeability of the cells. Then use normal sheep serum or BSA to block, so that many cell proteins first bind non-specifically to the blocking agent, and the specific target protein primary antibody can bind to the target protein through a competitive reaction, thereby ensuring the specificity of antibody recognition. Then, a fluorescent secondary antibody matching the species of the primary antibody can be used to specifically identify the Fc region of the primary antibody. Using the fluorescent group connected to the secondary antibody, a specific color of fluorescence can be observed under a fluorescence microscope, thereby showing the expression of the target gene. At the same time, immunofluorescence experiments can show the subcellular situation of gene expression (inside the nucleus, outside the nucleus, on the cell membrane and some organelles) due to its high sensitivity, so it is usually used as a method for locating the target protein.

Experimental purpose

Detect the expression and localization of the target protein in cells (or tissues).

Immunofluorescence detection method

(1)Direct immunofluorescence

Antibodies carrying fluorescein directly react with antigens to form antigen-fluorescein-labeled antibody complexes. The advantages of this method are that it is simpler and has higher specificity; the disadvantages are that it has a narrow range of application and lower sensitivity.

(2)Indirect immunofluorescence

First, a specific antibody is used to bind to the corresponding antigen in the cell, and then a fluorescein-labeled secondary antibody is used to bind to the specific antibody to form an antigen-specific antibody-labeled fluorescent antibody complex (the most commonly used method in the laboratory).

Introduction to indirect immunofluorescence

The main steps of indirect immunofluorescence experiments include sample preparation, fixation, permeabilization, blocking, incubation of primary and secondary antibodies, and final laser confocal photography.

1. Cell preparation

Cells are seeded in a 24-well plate with a slide, and the samples are processed when the cells grow to a suitable density. Too high a density will cause the cells to be too crowded, with poor morphology and unclear boundaries, and cause the staining background to be too dark. If there are too few cells, the cell activity will be poor, which will easily lead to non-specific staining. Therefore, the cell density should be around 60%-70% during staining.

2. Fixation

Fix with 4% PFA at room temperature for 30 min. The choice of fixative depends on the nature of the antigen being studied and the characteristics of the antibody used. Currently, 4% formaldehyde is more commonly used and is suitable for studying cell membrane proteins.

3. Permeabilization

After fixation, wash three times with 1×PBS, add 0.1% TritonX-100 100 μL/plate, and permeabilize at room temperature for 10 min. The purpose of permeabilization is to make holes in the cell membrane so that antibodies can enter the cell and bind to antigens.

4. Blocking

After permeabilization, wash three times with 1×PBS and add 10% FBS PBS 100 μL/piece for blocking. Blocking is to prevent endogenous nonspecific protein antigens from binding.

5. Primary antibody incubation

Dilute the corresponding primary antibody with 10% FBS PBS at a dilution ratio of 1:500, and add 100 μL/piece of diluted primary antibody. Incubate at room temperature for 1 h.

6. Secondary antibody incubation

After 1 h, wash three times with 1×PBS, and dilute the secondary antibody of the same species as the primary antibody with 10% FBS PBS at a dilution ratio of 1:500 or 1:1000. Add 100 μL/piece of diluted secondary antibody. Incubate at room temperature for 1 h.

7. DAPI nuclear staining

After the secondary antibody incubation, wash three times with 1×PBS, add DAPI 100 μL/slide, and stain the nucleus at room temperature for 10 min.

8. Sealing

Sealing: After the nuclear staining is completed, wash three times with 1×PBS, then wash three times with ultrapure water, add anti-quenching sealing agent on the slide, discard the water from the slide and gently invert it on the slide, dry it at room temperature away from light, and store it at -20℃.

Notes:

The primary antibody must be from a different species, and the species of the fluorescently labeled secondary antibody and the primary antibody must match, and the secondary antibody cannot have cross-reactions;

From the beginning of incubation of the secondary antibody, pay attention to light-proof operation, especially ultraviolet light irradiation, to avoid false negative results caused by fluorescence quenching;

During the washing process, the action should be gentle to prevent the cells from being blown away;

Select the appropriate cell density for the experiment.

Summary of common problems

1. Weak fluorescence signal

(1) Wrong excitation wavelength

Solution: Make sure the excitation wavelength matches the excitation wavelength of the fluorescent group

(2) Incompatible primary or secondary antibodies

Solution: Pay attention to species issues

(3) Over- or insufficient fixation

Solution: Adjust the time appropriately

(4) Improper sample storage

Solution: Pay attention to light protection

(5) Primary antibody is not easy to use

Solution: It is recommended to do a positive control to confirm the effectiveness of the antibody

2. High background

(1) Insufficient washing

Solution: Wash thoroughly and increase the number of washes appropriately

(2) Sample drying

Solution: Incubate the antibody in a wet box to prevent sample drying

(3) Insufficient blocking

Solution Solution: Extend the time or replace the blocking solution

(4) Antibody concentration is too high

Solution: Pre-experiment titration to confirm the optimal concentration

(5) Secondary antibody non-specific binding

Solution: Do not add primary antibody, only add secondary antibody as a control

(6) Sample autofluorescence

Solution: Detect sample autofluorescence in advance

3. Cell/tissue morphology is destroyed

(1) Tissue sections fall off the slide or there are bubbles in the sections

Solution: Increase the fixation time appropriately

(2) Tissue sections are torn or wrinkled

Solution: The cutting blade is not sharp enough, consider re-slicing

(3) Cells or tissues are not fixed sufficiently and spontaneously lyse

Solution: Increase the fixation time appropriately and use a cross-linking fixative