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Antibody common problems of IHC

Antibody common problems of IHC


The reason


Edge effect

The edge of the tissue is not firmly attached, and the edge tissue is loose and floating in the liquid

APES/polylysine treated glass slides; thin tissue sections as much as possible; try to avoid using tissues with more necrosis

The reagent does not cover the tissue adequately

When adding reagents, let the reagents completely cover the tissue

Non-specific chromosome

Antibody quality issues

Use quality-assured antibodies

Long antibody incubation time

Reduce incubation time

Antibody concentration is too high

Decrease antibody concentration

The primary antibody is polyclonal

Use monoclonal antibodies

Endogenous peroxidase and biotin

Extend inactivation time

Insufficient closure

Extend the closing time

DAB incubation time is too long or the concentration is too high

Configure reagents according to the instructions, and control the reaction time under the microscope

Insufficient washing after antibody incubation

Wash 3*5 times after each incubation

Dry tablets when reagent is dropped

Add reagents in time

Sections are soaked in buffer for too long

Block and add primary antibody in time after antigen retrieval, not overnight

Negative result

Antibody concentration and quality

Choose quality-guaranteed antibodies, do a preliminary experiment to find out the concentration of the antibody

Incomplete antigen retrieval

Explore the appropriate antigen retrieval method and time

Antigen abundance

Normal negative

Closed for too long

Reduce closing time

DAB incubation time is short

Control the color development time under the lens

Insufficiency of cell permeability, the antibody failed to enter the cell reaction

Choose the appropriate permeabilizing agent and its reaction time

Primary antibody and secondary antibody do not match

Choose the correct source of secondary antibodies

Take off

The slide is not processed

Clean the slides and treat them with APES or polylysine

Uneven slice thickness

Replace the blade; adjust the state of the slicer

Not enough baking time or temperature

60 food intake for 2 hours

Organizational problems

Tumor necrotic tissue and bone tissue itself are easy to fall off

Rapid temperature changes during antigen retrieval

Allow it to cool naturally after antigen retrieval

Excessive force during operation

Don’t shake the slices that are suspected of falling off, use absorbent paper to absorb the remaining liquid

Weak staining

Low antibody concentration and short incubation time

Increase antibody concentration and prolong reaction time

Reagent used beyond the expiration date

Reagent timing update

Too much residual buffer when adding reagents dilutes the reagents

Minimize residual liquid before adding reagents


Reduce closing time

Uneven coloring

Inconsistent slice thickness

Replace the blade; adjust the state of the slicer

The reagent preparation is not mixed and the local concentration is inconsistent

The reagents are mixed thoroughly and then added dropwise

The reagent does not completely cover the tissue

When adding reagents, let the reagents completely cover the tissue

Incomplete dewaxing

Replace dewaxing agent or extend dewaxing time

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