Principles and experimental procedures of immunohistochemistry
A principle of immunohistochemistry Immunohistochemistry uses the principle of specific binding of antigens and antibodies to determine the intracellular antigens of tissues by chromogenic reagents (fluorescein, enzymes, metal ions, isotopes) labeled with antibodies through chemical reactions ( Peptides and proteins), and its localization, qualitative and quantitative research is called immunohistochemistry.
2. The experimental procedure of immunohistochemistry is fixed. Immersion: Put the tissue sample directly into the fixative solution (4% paraformaldehyde and other fixative solution), soak at 4 ℃ for 2 hours, not more than 12 hours 2. Perfusion type: Mainly suitable for brain tissue, perfusion from the ventricle Normal saline evacuated blood from blood vessels and perfused with fixed solution slices
1. Paraffin section:
(1) Use ethanol from low concentration to high concentration to dehydrate the tissue;
(2) Immerse the tissue in xylene transparently;
(3) In the wax melting box, the tissue is embedded in paraffin;
(4) After cooling the embedded wax block, fix it on the microtome, cut into thin slices, and stick the thin slices on the glass slide;
(5) Dewax with xylene and soak ethanol from high to bottom again;
Note: After the paraffin section is prepared, it is necessary to perform antigen repair to unlock the amino group or carboxyl group on the epitope cross-linked by formaldehyde. Common methods include microwave heat repair, boiling heat repair, and enzyme digestion. 2. Frozen section: fixed The tissue was put into liquid nitrogen or dry ice-acetone to cool quickly, then sliced ​​by the microtome, and the piece was attached to the slide glass and sealed
1. To prevent peeling, you can use resin glue or polylysine for adhesion.
2. To avoid the influence of endogenous peroxidase, it can be treated with 3% hydrogen peroxide for 15min, (for antibodies labeled with peroxidase)
3. To avoid the influence of endogenous biotin, you can use egg white or egg white to seal;
4. Avoid non-specific staining, you can use secondary antibody-derived serum for blocking staining
1. Add the diluted primary antibody dropwise, wash at 4 ℃ overnight, wash with PBS or skim milk for 5min 3 times;
2. Add the diluted secondary antibody dropwise, incubate at 37 ℃ for 30min, wash with PBS or skim milk for 5min 3 times;
Color development: select the color development system matched with the secondary antibody and carry out the reaction. After the reaction is stopped by PBS, the cells are dehydrated with hematoxylin nuclei, ethanol gradient dehydration, xylene transparent, neutral gum sealer, dried at 37 ℃ for 48 hours under a microscope Observed
2. The experimental procedure of immunohistochemistry is fixed. Immersion: Put the tissue sample directly into the fixative solution (4% paraformaldehyde and other fixative solution), soak at 4 ℃ for 2 hours, not more than 12 hours 2. Perfusion type: Mainly suitable for brain tissue, perfusion from the ventricle Normal saline evacuated blood from blood vessels and perfused with fixed solution slices
1. Paraffin section:
(1) Use ethanol from low concentration to high concentration to dehydrate the tissue;
(2) Immerse the tissue in xylene transparently;
(3) In the wax melting box, the tissue is embedded in paraffin;
(4) After cooling the embedded wax block, fix it on the microtome, cut into thin slices, and stick the thin slices on the glass slide;
(5) Dewax with xylene and soak ethanol from high to bottom again;
Note: After the paraffin section is prepared, it is necessary to perform antigen repair to unlock the amino group or carboxyl group on the epitope cross-linked by formaldehyde. Common methods include microwave heat repair, boiling heat repair, and enzyme digestion. 2. Frozen section: fixed The tissue was put into liquid nitrogen or dry ice-acetone to cool quickly, then sliced ​​by the microtome, and the piece was attached to the slide glass and sealed
1. To prevent peeling, you can use resin glue or polylysine for adhesion.
2. To avoid the influence of endogenous peroxidase, it can be treated with 3% hydrogen peroxide for 15min, (for antibodies labeled with peroxidase)
3. To avoid the influence of endogenous biotin, you can use egg white or egg white to seal;
4. Avoid non-specific staining, you can use secondary antibody-derived serum for blocking staining
1. Add the diluted primary antibody dropwise, wash at 4 ℃ overnight, wash with PBS or skim milk for 5min 3 times;
2. Add the diluted secondary antibody dropwise, incubate at 37 ℃ for 30min, wash with PBS or skim milk for 5min 3 times;
Color development: select the color development system matched with the secondary antibody and carry out the reaction. After the reaction is stopped by PBS, the cells are dehydrated with hematoxylin nuclei, ethanol gradient dehydration, xylene transparent, neutral gum sealer, dried at 37 ℃ for 48 hours under a microscope Observed
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