1. What tissue and cell specimens are used in immunohistochemistry experiments?
The experimental use is mainly for tissue specimens and cell specimens, the former including paraffin sections (pathological large pieces and tissue chips) and frozen sections, the latter including tissue prints, cell crawling tablets and cell smears. Among them, paraffin sectioning is the most commonly used and basic method for making tissue specimens, which is well preserved for tissue morphology and can be continuously sliced, which is conducive to various staining control observations; it can also be archived for a long time for retrospective study; paraffin section production process has a certain impact on antigen exposure in tissues, but antigen repair can be carried out, which is the preferred tissue specimen production method in immunohistochemistry.
2. Why should paraffin sections do antigen repair? What are the methods?
Paraffin section specimens are fixed with formaldehyde, so that intracellular antigens form aldehyde bonds, carboxylic bonds and partial antigenic determinants are blocked, and crosslinks occur between proteins to conceal antigenic determinants. Therefore, it is required that when performing ihc staining, antigen repair or exposure needs to be carried out first, that is, the crosslinking formed between the molecules when fixed is destroyed, and the original spatial form of the antigen is restored. Commonly used antigen repair methods are microwave repair method, high pressure heating method, enzyme digestion method, boiling heating method, etc., and the commonly used repair solution is 0.01 mol/l citrate buffer of ph6.0.
3. What are the commonly used staining methods for immunohistochemistry?
According to the different markers, it is divided into immunofluorescence method, immuno-microplypease method, affinity histochemistry, the latter is a detection method based on a substance with a high affinity for a certain tissue component. This method is more sensitive and facilitates the localization of trace antigens (antibodies) at the cellular or subcellular level, of which biotin-antibiotin staining is the most commonly used.
4. Paraffin sections are desped from the tablets during the dyeing process
1) The baking time is not enough, or the temperature is not enough, you can extend the baking time and increase the baking temperature;
2) With slides containing polylysine, you can buy it or do it yourself;
3) Some tissues themselves are easy to fall off, such as bone tissue, etc., when operating, punch pbs do not rush directly to the tissue, rush to the top of the tissue, let it flow down to flush the tissue;
4) When repaired with high temperature, sudden temperature cold may also be caused.
5. Edge effect
1) The edge of the tissue and the slide are not firmly pasted, the edge tissue is loose and floating in the liquid, and it is not easy to wash away the reagent under the tissue each cleaning. Solution: Prepare high-quality film (apes or polylysine), cut out as thin as possible tissue sections, not thicker than 4 microns, the pretreatment of tissue should be standardized, try to avoid the use of necrotic tissue;
2) The reagents added dropwise on the slices do not adequately cover the tissue, and the reagents at the edges are easy to dry out first, and the concentration is higher than that of the central tissue and causes staining depth. Solution: To adequately cover the tissue, the reagent should exceed the edge of the tissue by 2 mm. When drawing circles with a grouping pen, in order to avoid the influence of oil agents, the circle should be 3-4 mm from the edge of the tissue.
6. Non-specific staining of tissue sections is generated
1) The antibody incubation time is too long and the antibody concentration is high, which is easy to increase the background coloring. This can be controlled by shortening the primary/secondary antibody incubation time and diluting the antibody. This is the most important one;
2) Polyclonal antibodies with primary antibodies are prone to non-specific coloring, it is recommended to try monoclonal antibodies to see;
3) Endogenous peroxidase and biotin are high in tissues such as liver and kidney (tissues containing many blood cells), and background staining needs to be reduced by prolonging inactivation time and increasing inactivator concentration;
4) Non-specific components bind to antibodies, which need to strengthen the blocking effect by prolonging the isolation time of animal immune serums of secondary antibody sources and appropriately increasing the concentration;
5) Dab incubation time is too long or the concentration is too high;
6) PBS irrigation is not sufficient, residual antibody results enhance coloring, in the primary antibody, secondary antibody or sp incubation after the dip is particularly important;
7) Dry tablets often appear during specimen staining, which easily enhances non-specific coloring.
7. Immunohistochemical staining showed negative results
1) Antibody concentration and quality problems and the wrong choice of antibody source;
2) Antigen repair is incomplete, for formaldehyde-fixed tissues must be repaired with sufficient antigens to open the epitope of the antigen to facilitate binding to the antibody; it is recommended that microwave repair be tried with high heat 4 times * 6min. Someone has done experiments, and this is the best time and number of times. If not, it can also be repaired by high pressure;
3) The antigen content of the tissue slice itself is low;
4) The serum is blocked for too long;
5) Dab incubation time is too short;
6) The cell permeability is incomplete, and the antibody fails to enter the cell sufficiently to participate in the reaction;
7) Start doing immunohistochemistry, I suggest you must first do a positive photo, exclude antibodies and other method problems.
8. Background
1) Consider high primary antibody concentration;
2) Then adjust the dab incubation time;
3) Also consider whether the serum blocking time is too short;
4) Appropriately increase the number of dips after antibody incubation and extend the dip time.
Source: The State of Cells