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Troubleshooting Tips

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 #11011.

 

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The antibody is not active Don’t use out-of-date antibody kits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 #11583.

 

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#11221.

 

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#11115.

 

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