Immunofluorescence of frozen sections
Immunofluorescence of frozen sections
Observation of localization of target proteins in tissues
Principle
Appliance immunostaining Operation method Immunofluorescence of frozen sections Materials and Instruments 【Materials】Tissue sections, primary antibody, fluorescence labeled secondary antibody; Move 1. Frozen sections are usually made on polylysine-adhesive slides. Polylysine-treated slides are strongly cationic and are mainly used for pathological tissue sections and liquid-based cytology smears to prevent slides from falling off. If you do not want to stain the slides immediately after freezing, you can place them in a negative 80 refrigerator. After taking them out again, they can be dried at room temperature for 15 minutes. Then soak in PBS for 10 minutes to remove OCT. 2; 2. the tissue to be stained is circled with a histochemical grease pencil in order to minimize the area for efficient incubation of the antibody when performing subsequent incubations; 3. 0.5% TritonX-100 (PBS prepared) permeabilized at room temperature for 20 min (this step is omitted for antigens expressed on the cell membrane); the sections are closed with PBS containing 10% normal goat serum at room temperature for 1 hour (this step can be done without washing, and the sealing solution can be drained off). The purpose of the sealing is to eliminate non-specific staining through the binding of serum to the nonspecific site, so as to improve the accuracy of the target protein and reduce the background. The purpose of containment is to eliminate non-specific staining by binding the serum to the non-specific site, to improve the accuracy of the target protein and to reduce background; 4. Add primary antibody (1:100-1:500) and incubate at 4°C overnight. The concentration of primary antibody can use the recommended concentration of antibody, or you can choose the appropriate concentration according to the quality of antibody. 5; 5. Wash with PBS 3 times for 10 minutes each time. 6; 6. Add fluorescently labeled secondary antibody (1:200) and incubate at 37℃ for 1 hour, taking care to avoid light. Commonly used fluorescein are FITC (bright yellow-green fluorescence), rhodamine (bright orange-red fluorescence). 7; 7. Wash with PBS 3 times for 10 minutes each time. 8; 8. Dilute DEPI (1:1000) with sheep serum and incubate for 5 min, protected from light, for the purpose of staining cell nuclei for 10 min. In addition to DEPI for nuclear staining, Hoechst staining can also be used, which generally fluoresces blue. 9; 9. Wash with PBS 3 times for 10 min each time. 10; 10. seal the slides with drops of antifluorescence quencher. A tip for sealing the slides is to put nail polish around the slides, which can effectively bond the coverslips to the slides. After sealing, the results were observed by fluorescence microscope and photographed. Caveat (1) Do not allow the surface to dry throughout the experiment.(2) Avoid light during dilution, addition of secondary antibody (fluorescent antibody) and subsequent washing.(3) Frozen sections need to be photographed as soon as possible after immunofluorescence to prevent immunofluorescence quenching.(4) When taking pictures by fluorescence microscope, choose the appropriate excitation light source according to the fluorescent antibody. For more product details, please visit Aladdin Scientific website.
[Reagents] Fixative, gradient sucrose, phosphate buffer, sealing solution, DAPI, fluorescence anti-quenching sealer;
[Apparatus] Frozen section machine, fluorescence microscope or laser confocal microscope;