Antibody common problems of IHC

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Antibody common problems of IHC

Problem	The reason	solution
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
Edge effect	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
	Over-closure	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

Antibody common problems of IHC

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