Troubleshooting tips for IHC

 Troubleshooting tips for IHC common problems:

1. Non-specifc staining

2. No staining

3. Weak staining 

4. Strong Staining 

 

1.Non-specifc staining

 

Causes	Solutions
Improper preparation of sections	Improve ways of sampling and preparation
Inadequate deparaffinization of the sections	Increase the deparaffinization time
Tissue contains endogenous peroxidase	Use 0.3% v/v fresh H2O2 for blocking and increase the blocking incubation time
Tissue contains endogenous biotin	Use IHC biotin blocking agent
Blocking of protein may be insufficient	Increase the blocking time
Charge adsorption	Block with nonimmune animal serum
The antibody is not pure	Change suitable antibody
Primary antibody concentration may be too high	Try decreasing the antibody concentration
The sections have dried out	Avoid sections being dried out in the process of experiments
Washes may be insufficient	Increase the times of washes and the washing time

 

Non-specifc staining:	Improved:

         

           

Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue showing cytoplasmic and nuclear staining using NFκB-p65 Phospho-Ser276 Antibody #AB11011.

 

2.No staining

Causes	Solutions
Improper tissue processing	Try to improve the condition and sampling again
No antigen in the tissue	Set a positive control to verify the experiment results
The antibody is not active	Don’t use out-of-date antibody kits
Incompatible secondary and primary antibodies	Use secondary antibody that was raised against the species in which the primary was raised
Incompatible staining system	Change compatible staining system
Improper operation and leave out important steps	Follow strict operating procedure and set a positive control

  

No staining:	Improved:

  

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using Histone H3 Di-Methyl-Lys27 Antibody #AB11583.

 

3.Weak staining 

Causes	Solutions
Improper tissue fixation or too high temperature when fixing	Use appropriate fixation way or fixation time
Too high baking slides temperature and too long baking time	Choose appropriate temperature and time for baking slides
The antigen may be damaged	Let fresh tissues be fixed in time and for not to exceed 24 hours
Over blocking of protein	Reduce the blocking time
The antibody has drained away	Ensure that the sections are placed in a horizontal position  when incubating
The antibody concentration may be too low or incubation time may be too short	Increase the antibody concentration and incubation time
The room temperature may be too low. Lower than 15℃.	Incubator at 37℃ or increase incubation time
No draining off buffer solution when adding the reagent results in the reagent being diluted.	Do drain off buffer solution but avoid sections being dried out.
Excessive washing	Wash moderately
Always verify the expiration date of the reagent prior to use	Change reagents timely

 

Weak staining:	Improved:

  

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using mTOR Phospho-Ser2448 Antibody#AB11221.

 

4.Strong Staining

 

Causes	Solutions
The primary antibody concentration may be too high or incubation time may be too long	Reduce the primary antibody concentration or incubation time.
The incubation temperature may be too high	Incubate at 4℃ or at room temperature
The incubation time of HRP conjugated secondary antibody may be too long	Reduce the incubation time
Inadequate washing	Increase the times of washing

 

Strong Staining:	Improved:

 

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using FKHR Phospho-Ser256 Antibody#AB11115.