I. Principle of Immunofluorescence

 

Immunofluorescence (IF) technology is based on the principle of antigen-antibody reactions. Known antigens or antibodies are first labeled with fluorophores to create fluorescent antibodies. These antibodies (or antigens) are then used as probes to detect corresponding antigens (or antibodies) within tissues or cells. The antigen-antibody complexes formed within tissues or cells contain labeled fluorophores. By observing the specimen under a fluorescence microscope, the fluorophores emit bright fluorescence (yellow-green or orange-red) when excited by external light, allowing visualization of the tissue or cells containing fluorescence. This helps to determine the nature and localization of antigens or antibodies and, using quantitative techniques, to measure their content.

 

II. Classification of IF

 

Chinese Full Name	Abbreviation	Chinese Full Term	Experimental Sample
Cellular Immunofluorescence	IF-IC	Cellular Immunofluorescence	Suspension or adherent cells
Tissue Immunofluorescence	IF-F	Frozen Section Immunofluorescence	Frozen tissues, better antigen preservation, but avoid ice crystal formation
	IF-P	Paraffin Section Immunofluorescence	Paraffin-embedded cell clusters or tissues, easy to store, good morphology

 

III.IF Experimental Procedure

 

1) Sample Preparation: For cells growing as a monolayer, when subculturing, seed cells into culture dishes pre-placed with treated cover slips. After the cells have grown into a monolayer, remove the cover slips and wash twice with PBS; For suspension cells, take cells in logarithmic growth phase, centrifuge and wash with PBS (1000rpm, 5min) twice, prepare cell smears using a cytocentrifuge or directly prepare cell smears;

2) Fixation: Fix cells with 4% Paraformaldehyde (PFA) solution for 15min, wash with PBS three times, 5min each;

3) Permeabilization: 0.5% Triton X-100, permeabilize for 15-20min, wash with PBS three times, 5min each;

4) Blocking: Block with 3% Bovine Serum Albumin (BSA) at room temperature for 30min-1h. The purpose of blocking is to reduce non-specific binding of primary and secondary antibodies to non-target sites, thereby reducing background signals;

5) Primary Antibody Incubation: After blocking, do not wash, directly aspirate the excess BSA, and incubate overnight at 4°C with diluted primary antibody (diluted in 3% BSA);

6) Secondary Antibody Incubation: Wash with PBST three times, 5min each, to remove unbound antibodies. Then incubate with diluted secondary antibody (diluted in 3% BSA) at room temperature in the dark for 1h, wash with PBST three times, 5min each;

7) Nuclear Staining: Add DAPI and stain in the dark for 5min, wash with PBS three times, 5min each (nuclear staining is for cell localization);

8) Imaging: Immediately capture images using a confocal or fluorescence microscope. If delay is needed, store in the dark at 4°C.

 

IV.Detection Methods

 

Detection methods are divided into direct and indirect tests. We generally use the indirect method (primary antibody + secondary antibody).

 

Antigen Detection	Directly labeled primary antibody	Directly labeled secondary antibody	Secondary antibody conjugated to HRP, tyramide signal amplification system
Advantages	Simple operation, one-step method, moderate signal strength not limited by antibody source when multiple staining is required	Moderate signal strength	Strong signal, not limited by antibody source when multiple staining is required
Disadvantages	Low signal strength	Two-step method, background may increase	Requires optimization, may introduce background or non-specific staining

 

V.Immunofluorescence Double Staining

 

If two antigens need to be detected simultaneously in the same tissue or cell specimen, dual fluorescence staining is required. Dual immunofluorescence labeling also includes direct and indirect methods.

1) Direct Method: Mix two different fluorophore-conjugated antibodies (e.g., anti-A and anti-B) in appropriate proportions, incubate with the sample, rinse, mount, and examine under a microscope;

Characteristics: Simple and reliable, no need to consider the species origin of primary antibodies, but lower sensitivity.

2) Indirect Method: Incubate the sample with two unlabeled primary antibodies, rinse, then add two different fluorophore-conjugated corresponding secondary antibodies, rinse, mount, and examine under a microscope;

3) Characteristics: When using this method, it is important to ensure that the two specific primary antibodies are from different species, and the species of the fluorophore-labeled secondary antibodies and primary antibodies must match.

 

VI.IF Precautions

 

1) Fixation time affects fluorescence fixation effectiveness. For cell samples, if fixed with 4% Paraformaldehyde, a recommended fixation time of 15min is suggested. Too short a time will not achieve the desired fixation effect, and too long will cause the formaldehyde to oxidize into formic acid, losing its fixation effect;

2) Keep aldehyde fixatives fresh, otherwise spontaneous fluorescence background will increase;

3) When using antibodies, consult the instruction manual. Since different antibodies have different usage scenarios, confirm whether the antibody can be used for cellular IF, frozen section IF, or paraffin section IF;

4) Cell density is one of the key points for the success of the entire experiment. If there are too many cells, overlapping can also affect the overall fluorescence effect;

5) Gently wash to prevent excessive force during the liquid addition process from causing cell loss;

6) The permeabilization time is generally 15-20min. Both too short and too long durations will not yield good results, and different time gradients should be explored.

 

VII.Summary of Common IF Issues

 

1) Weak or no signal

Possible Cause	Solution
The target protein requires induced expression or has low expression levels	For proteins requiring induced expression, refer to literature to find the best conditions for inducing protein expression and positive controls; for proteins with low expression levels, consider signal amplification or pairing with brighter fluorophores; whenever possible, use Western blotting or other methods to confirm protein expression
Improper sample storage. For example, exposure to light for too long may cause signal decay	Incubate and store samples under light-protected conditions. Seal samples in quenching solution. Immediately capture images after sealing to obtain the best results
Samples have been stored for too long	Use freshly prepared sample slides/plates
Insufficient fixation	Refer to the product manual; immediately remove the medium and thoroughly wash in the fixative after treatment. For phospho-specific antibodies, use a formaldehyde concentration of at least 4% to inhibit endogenous phosphatases
Incorrect antibody dosage	Refer to the recommended antibody dilution concentrations in the manual and optimize based on protein expression levels
Incorrect primary antibody incubation time	It is recommended to incubate overnight at 4°C for the best results
Incorrect permeabilization method	Refer to the recommended operating steps in the product manual
Improper secondary antibody usage	Use at the recommended concentration and check if the secondary antibody matches the species of the primary antibody
Incorrect excitation wavelength	Ensure that the excitation and detection (laser/excitation/emission filters) match the excitation wavelength of the fluorophore

 

2) High background

Cause	Solution
Insufficient blocking	Use normal serum from the same species as the secondary antibody for blocking for 1 hour
Antibody concentration is too high	Refer to the recommended concentration in the manual and optimize based on protein expression levels
Antibody incubation time is too long or incubation temperature is too high	Refer to the recommended incubation time and temperature in the manual
Sample has dried	Ensure that the sample is always completely submerged in liquid during the staining process
Insufficient washing	Wash to remove excess fixatives, excess secondary antibodies, and loosely bound, non-specific antibody interactions
Sample autofluorescence	Use an unstained sample as a control to detect the degree of autofluorescence. Refer to the fixatives recommended in the manual; old fixatives may exhibit autofluorescence. Replace old formaldehyde and prepare fresh dilutions. Use freshly diluted glutaraldehyde in ampoules that can be used for electron microscopy (EM grade). Choose longer wavelength channels for low-abundance targets
Non-specific antibody binding	If possible, compare with cells knocked down (siRNA) or negative cells, or compare with cells known to have high or low levels of target antigen expression

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us

Absin Bioscience Inc. Email: worldwide@absin.cn

Follow us on Facebook: Absin Bio