Immunofluorescence dyeing experiment

Immunofluorescence dyeing experiment

Immunofluorescence is one of the early development of immunization technology, it is in immunology, biochemistry and A technique established on the basis of microscope technology, by combining antibodies with some tracer substances, the positioning of tissue or intracellular antigens is carried out using antigenic antibody.
1, cell fixation and transparent

In order to achieve better detection effect, cells need to be fixed and transmitted. These steps are very critical, cells and antigens need to be structurally structurally, and are beneficial to antibodies and antigen binding. Normally, it is necessary to achieve the following steps: cells are fixed with 2% -4% polymethylene formaldehyde, followed by 0.1% saponin or 0.02% Triton X-100. The former is a relatively gentle process, but the probability of the nuclear antigen may be invalid, Triton is required. When using saponin, it is important to pay attention to it will cause reversibility of the cell membrane, that is, in addition to the initial introduction, there is a need for transmission in each antibody incubation. In addition, the cells can be fixed and transmitted simultaneously with ice alcohol, which can avoid the use of the detergent.

2, antibody-specific

Immunofluorescence requires a very strong antibody to avoid high background and unsatisfactory protein positioning results. In most cases, the effect of purifying the antibody is very good, but the correct control can help localize the antigen. The use of only two anti-dyeing films is used as a negative control, which is advantageous to reduce reducing background interference.

3, suitable antibody dilution ratio

Optimize dyeing, usually 1 ug / ml purified antibody or 1: 100-1: 1000 anti-precision is sufficiently achieved Specific dyeing results. However, in the premise of low background staining, the signal strength can be improved by adding concentrations. The concentration gradient experiment is highly recommended if it is * times using the antibody or determined an antigen.

4, optimized buffer and blockage

Although many antigen can be dyed in common buffers such as PBS, for some object of antigen, replace it contains different ions. Buffer, such as calcium, magnesium, potassium, etc., can have a large extent. Rockland provides optimized IHC with closed buffer, equally applicable to fluorescent dyeing experiments.

5, choose the correct secondary anti

If you need to do immunization experiments, we strongly recommend that you choose to perform a secondary anti-interpolation of secondary; if it is double or even It is multiple dyeing, then a pre-ad. is used. At the same time, please prefer the secondary antique

Immunofluorescence dyeing experiment The customer provides

Cell strain or cell climbing.Note: Cell climbing should not be too dense; cells are fixed.Slices of tissue, one-to-art anti-

Basic experimental steps, drugs, sample processing, pictures and picture analysis, fluorescence microscopy or confocalMicroscope shooting

Experiment cycle: 1-3 weeks