8 pitfalls and troubleshooting in IHC

Firstly, let’s see 8 regular problems in IHC and then the quick troubleshooting list.

1. Trouble in the primary antibody
It is more than important to choose the purified antibody which has high titer and specificity. Monoclonal antibody works better in IHC which causes less non-specific background. Check our best cost-effective Monoclonal antibody for IHC.

2. Concentration of antibody is too high/ Excessive incubation of antibody
Concentration of primary antibody is crucial in the IHC. Sometimes, we can’t simply follow the datasheet of antibody but performing preliminary test to determine the better concentration. Meanwhile, to make sure time of incubation is in control, a TIMER is very helpful. After adding antibody to tissues, set the timer immediately for accurate time control.

3. DAB staining lasts too long
Time of DAB staining is different case by case. We need to monitor the process under microscope every time. Once light brown color appears in the tissues, we need to wash the slides immediately. If seeing brown color too fast, antibody concentration is too high; If it takes a long time to see the brownness, antibody concentration is too low.
It is better to use fresh DAB and keep it in dark and dry condition. Do not add H2O2 to DAB until it is ready for use.


8 pitfalls and troubleshooting for IHC. AntibodyPlus
Figure.1 Staining lasts too long in left picture and it should be washed earlier. Timing of right picture is perfect.

4. Endogenous biotin or enzyme
Many organs contain abundant endogenous biotin or enzyme, such as liver, kidney and spleen. It is very easy to cause non-specific staining if not handled appropriately.
To deactivate most of alkaline phosphatase, we suggest adding Levamisole (24 mg/ml) into substrate and keep pH around 7.6-8.2.
For acidic phosphatase, 50 mmol/l tartaric acid could inhibit their activity.
To inhibit endogenous peroxidase, 0.9% H2O2 is commonly used.
To inhibit endogenous biotin, before staining, rinse slides in 25 ug/ml Avidin for 15 mins and wash slides with PBS for another 15 mins.

5. Tissue becomes dry
Don’t make more than 4 slides for IHC at the same time.
Let all the reagents cover the tissues completely (extent for 2mm outside the edge of tissue).
Draw a circle around the tissue using PAP pen (circle should be 3-4 mm outside the edge of tissue). It could prevent loss of reagents applied.

6. Rinse slide in buffer overnight
Don’t rinse slides too long in buffer or in antigen retrieval buffer. If you have to leave them in buffer overnight due to time arrangement, put them in 4 Celsius degrees and then leaving for overnight should be fine.

7. Insufficient clearance
It is important to clear slides completely using PBS. PBS should be prepared using ddH2O and it needs to be re-validated for pH before using. Use 0.05 mol/L Tris-HCl and 0.15 mol/L NaCl for Buffer is good enough. It is better to add some Tween-20 as instructed.

8. Blocking buffer
Use the correct serum in blocking buffer. Principle is choosing the non-immune serum from secondary antibody host species.
Dilute to 3-10% solution using PBS (37 Celsius degrees 10-30 mins). After blocking, we could directly shake the slides to dry. There is also a good way to use 5% non-fat milk for blocking.

Secondly, let’s see four problematic results which are commonly observed.
This is aimed for your quick troubleshooting.

1. All slides show negative results.
Possible reasons
1.1 Staining is not performed as instructed
1.2 primary or secondary antibody is not added
1.3 There may be NaN3 in buffer which inhabits activity of enzymes
1.4 H2O2 in substrate is inadequate or lost effective
1.5 inappropriate counterstain or dehydration before detection

2. All slides show positive results
Possible reasons
2.1 Slides are too dry or antibody is too concentrated in the slides
2.2 NaCl isn’t added in the buffer and pH of buffer is inappropriate
2.3 Staining lasts too long
2.4 Incubation of antibody lasts too long
2.5 Concentration of H2O2 is too high; process of staining is too fast

3. High background
Possible reasons
3.1 Endogenous peroxidases are not blocked
3.2 Slides are too thick
3.3 Insufficient time of rinse
3.4 Staining lasts too long
3.5 Incomplete blocking of protein; Hemolysis in serum
3.6 Not enough dilution of antibody

4. Uneven dyeing
Possible reasons
4.1 Deparaffinization and rehydration is insufficient
4.2 Antibody isn’t mixed well
4.3 During incubation of antibody, slides are tilting
4.4 After incubation of antibody, PBS is not cleared
4.5 Thickness of slide is uneven