Immunohistochemistry technical issues

In the process of immunohistochemistry, in order to better explain the problem and find the reason, it is best to set a positive pair of photos (known tissues that highly express the corresponding antigen) and a negative control tissue piece (no primary antibody but secondary antibody system / Without any antibody (two groups), all operations should be exactly the same.

Weak staining or no staining (in order of priority, some simple reasons are excluded first):

If the reagents are used in the wrong order or if the reagents are omitted, repeat the experiment to ensure that each step is correct. The secondary antibody and the primary antibody do not match. Choose the matching secondary antibody substrate and enzyme. Mix with substrate to see if it develops color?) There is no test antigen expression in the tissue or the expression level is low. Use a positive antibody antibody concentration that is too low to increase the antibody concentration. It is best to use different concentrations of antibodies to determine the optimal concentration of antibody incubation time. Fully extend the antibody incubation time. Substrate incubation is insufficient. Improper storage of the substrate incubation time leads to improper antibody storage, resulting in failure. Separate the antibody and store it at low temperature (-20 to -70 C) to avoid repeated freeze-thaw. Dilute the antibody to store for 1-2 weeks at 4 to avoid too long. Or according to the instructions to properly preserve the failure of the primary antibody and replace it with a new primary antibody secondary antibody and replace with a new antibody (can it be successfully combined with other primary antibodies?) Deparaffin does not extend the dewaxing time sufficiently or replace with a new secondary antibody Toluene tissue fixation is inadequate or improperly increases the fixation time or changes to a different fixation method to fix the tissue excessively and reduce the fixation time. If it has been fixed too much, you need to use the correct or recommended antigen repair method. The counterstaining reagent does not match the substrate system. Select the correct counterstaining reagent (without counterstaining, see if there is tissue staining?) The tablet is not sealed correctly Choose the right tablet

High staining background:

Insufficient film washing. At least 3 times between each step. The tissue contains endogenous enzymes, such as HRP / AP. Before adding the primary antibody, use 3% H2O2 methanol to block the endogenous HRP activity. Block endogenous AP activity. Or switch to glucose oxidase system tissue containing endogenous biotin (especially kidney, liver, and spleen). Before adding the primary antibody, use avidin / biotin blocking reagent after serum blocking. Or avoid using the biotin-avidin system or switch to the IF method (add enzyme and substrate directly to the tissue slice to see if it develops color?) Non-specific binding of the primary antibody or excessive antibody concentration increases the dilution of the primary antibody Non-specific tissue binding is blocked with normal animal serum (usually 10%) from the same source as the secondary antibody. If necessary, the serum concentration can be increased and tissue fixation is insufficient. Antigen diffusion Increase duration of postfixation. Avoid staining of mouse tissue before adding primary antibody with MouseOnMouse blocking reagent Too high primary or secondary antibody concentration reduces antibody concentration. Or use different concentrations to determine the optimal staining concentration. Incubation time is too long. Incubation time is too high. Incubation temperature is too high to reduce incubation temperature. Overnight at 4 ℃ or 37 ℃ 0.5-1 hour or RT substrate incubation time is too long. Avoid dry flakes during the dyeing process

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