Generic Troubleshooting Guide for IF/ICC and IHC

1. No staining


Improvement Suggestion

The primary antibody and the secondary antibody are not compatible.

Use a secondary antibody that was raised against the species in which the primary was raised (e.g primary is raised in rabbit, use anti-rabbit secondary).

Not enough primary antibody is bound to the protein of interest.

Increase the concentration of primary or secondary antibodies. Incubate for longer periods of time (e.g. overnight) at 4oC.

The antibody may not be suitable for IHC procedures which reveal the protein in its native (3D form).

Test the antibody in a native (non-denatured) WB to ensure this is recognized.

The primary/secondary antibody/amplification kit may have lost its activity due to improper storage, improper dilution or extensive freezing/thawing.

Run positive controls to ensure that the primary/secondary antibody is working properly.

The protein is not present in the tissue of interest.

Run a positive control recommended by the supplier of the antibody.

The protein of interest is not abundantly present in the tissue.

Use an amplification step to maximize the signal.

The secondary antibody was not stored in the dark.

Always prevent the secondary antibody from exposure to light.

Deparaffinization may be insufficient.

Deparaffinize sections longer, change the xylene.

Fixation procedures may be masking the epitope.

Use antigen retrieval methods to unmask the epitope. Fix for less time. Under fixation can cause heavy edge staining, but no signal at the centre of your specimen. See our fixation tips for further information.

The protein is located in the nucleus and the antibody cannot reach it.

Add a permeabilizing agent to the washing buffer and/or antibody dilution buffer. This can be Tween 20, but Triton X100 is stronger. The concentration depends on the thickness of your section.

The PBS buffer is contaminated with bacteria that damage the phosphate groups on the protein of interest.

Add 0.01% azide to the PBS antibody storage buffer or use fresh sterile PBS.

The exposure time is too short.

Increase the time of exposure for capturing the signal.

2. Weak Staining


Improvement Suggestion

The antigen may be damaged

Fix fresh tissues within 24 hours.

Excess buffer solution when adding the reagent resulting in the reagent being diluted.

Drain off buffer solution however, avoid sections drying out by keeping in high humidity.

Excessive washing.

Wash moderately.

The antibody has drained away.

Ensure that the sections are placed in a horizontal position during incubation.

Improper tissue fixation and/or too high temperature when fixing sample.

Use appropriate fixation method and/or fixation time.

Check the reagents do not exceed their expiration dates.

Change reagents before the expiration date arrives.

Baking slides in too high a temperature and/or for too long a time.

Choose appropriate temperature and time for baking slides.

Over blocking of protein.

Reduce the blocking time.

The incubation time may be too short and/or the antibody concentration may be too low.

Increase the incubation time and/or antibody concentration.

The room temperature may be too low, ensure the temperature is not lower than 15℃.

Incubate at 37℃ or increase incubation time.

3. High Background


Improvement Suggestion

Blocking of non-specific binding might be absent or insufficient.

Increase the incubation time and/or the concentration of the serum in the blocking buffer. Change the blocking agent. Biorbyt recommends blocking with 10% normal serum for 1 hr for sections or with 1-5% BSA for 30 min for cells in culture.

The primary antibody concentration may be too high.

Lower the concentration of your primary antibody.

Incubation temperature may be too high.

Incubate sections or cells at 4°C.

The secondary antibody may be binding non specifically (damaged).

Run a secondary control without primary antibody.

Fixative still present.

Wash extensively with TBS or PBS between all steps.

Endogenous peroxidases are active.

Use enzyme inhibitors (e.g. Levamisol (2 mM) for alkaline phosphatase or H2O2 (0.3-3% v/v) for peroxidase).

Fixation procedures are too strong and modified the epitope that the antibody recognizes.

Change antigen retrieval method, decrease the incubation time with the antigen unmasking solution.

Too much amplification.

Reduce the amplification incubation time and dilute the amplification kit.

Too much substrate was applied (enzymatic detection).

Reduce substrate concentration and incubation time.

The chromogen reacts with the PBS present in the cells/tissue (enzymatic detection).

Use TBS buffer to wash sections prior to incubating with the substrate. Then wash sections/cells in TBS buffer again after substrate incubation.

Pemeabilization has damaged the membrane and removed the membrane protein.

Remove permeabilizing agent from your buffers.

4. Non-Specific Staining


Improvement Suggestion

Primary/secondary antibody concentration may be too high.

Try decreasing the antibody concentration and/or the incubation period. Compare signal intensity against cells that do not express the target.

Endogenous peroxidases are active.

Use enzyme inhibitors (e.g. Levamisol (2 mM) for alkaline phosphatase or H2O2 (0.3-3% v/v) for peroxidase).

The primary antibody is raised against the same species as the tissue stained (e.g. mouse primary antibody tested on mouse tissue). When the secondary antibody is applied, it binds to all the tissue as it is raised against that species.

Use a primary antibody raised against a different species than your tissue.

The sections/cells have dried out.

Keep sections/cells at high humidity to avoid them drying out.