Shanghai immunofluorescence dyeing experiment

Immunofluorescence dyeing experiment Immunofluorescent cell chemistry is based on antigen antibodies The principle of the reaction, first form a known antigen or antibody labeled fluorescein, and then use such a fluorescent antibody (or antigen) as a molecular probe to inspect the cell or tissue within the cell or tissue. The antigenic antibody complex formed in the cell or tissue contains fluorescein, and the specimen is observed by a fluorescent microscope, and a bright fluorescence (yellow-green or orange) can be seen by the illumination of the fluorescent microscope. To determine the nature, positioning, and use of quantitative techniques for determining the content of the antigen or antibody.

Second, Shanghai Immunofluorescent dyeing experiment

Will not affect antigenic antibody activity fluorescent pigment markers Antibody (or antigen), after the corresponding antigen (or antibody) thereof, a specific fluorescence reaction is presented under a fluorescent microscope. Third, Shanghai

Immunofluorescence dyeing experiment

Immunofluorescence list mark

Immunofluorescence monomer refers to only one protein molecule, and the method is relatively simple, as long as it is done according to the dyeing step, there is usually no problem. However, pay attention to the choice of fixed liquid, the appropriate or not, the appropriate or not, may directly affect the dye results. The specific dyeing method is as follows. 1, the desired material and reagent

A, the degree of fusion in the cover slide or glass culture dish reaches 60% -70 %Cell.

B, Anti-anti-Fitc or Tritc labeled secondary antibody.

C, 4% polymethylene fixed liquid or cold acetone fixation (20 min is 20 min).

D, the sealing liquid.

E, 0.01 mol / lpbs buffer.

2, dyeing method

A, take out the cultured cover slide or Glass-Bottom culture dishes, wash 2 with 0.01 Mpbs 2 -3.

B, add 0.3% Tritonx-100, 37 ° C, 30 rain

CFurther, 4% polymethylene formaldehyde was added to fix 30 min or cold acetone at 4 ° C for 10 min. D, normal blocking serum 1: 20 closed trial 20-30 min, inhibiting non-specific binding of IgG. Block serum selection and normal serum of the secondary anti-same belong.

E, remove normal serum, add directly to the primary antibody, incubate at 37 ° C for 1 h or 4 ° C overnight. The antibody was diluted at 0.01 Mo / LPBS (the desired antibody concentration required to be trial). F, 0.3% Tritonx-100 washing 5 min, 0.01 mol / ipbs wash 5 min, 0.3% Tritonx 5 min, 0.01 m PBS wash 5 RAIN.

g, add FITC-borne or Tritc-secondary, 37 ° C, incubation for 1 h. H, 0.3% Tritonx-100 washing 5 mil, 0.01 mol / lpbs wash 5 min, 0.3% Triton x 5min, 0.01 mol / lpbs wash 5 min, and dry it with filter paper.

I, 90% glycerol (PBS formulation) seal. J, observation under laser scanning confocal microscope, or preserved in 4 ° C.

The immunofluorescence double marker refers to two protein molecules in the cells in the same manner, when doubting some kind The body can be proven to be used after the body is binding. This method is slightly more complex, first-should be used to pay attention to the two-specific antibodies used from different animals (eg, antibodies as polyclonal antibodies, from rabbits; B antibodies are monoclonal antibodies, from mice) . Second, the emitting light of the two secondary antibolibins should not overlap, and as far as possible, FITC and TRITC, ALEX488, and TRITC, FITC, and CY5, or CY3 and CY5, etc. can be selected. Under normal circumstances, two primary antibody can be incubated simultaneously, and then incubate both two kinds of bilateral. However, when the result of dyeing is very weak, and when another color is strong, it should be considered first-to-incubation of two-resistance. Other steps are marked with immunocemic fluorescence.

Fourth, customers provide

Cell strain or cell climbing. Note: Cell climbing should not be too dense; cells are fixed. Tissue sections, one-to-arm

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

Experiment cycle: 1-3 weeks