Suboptimal IHC staining is frequently resolved by adjusting relatively few variables. Adjustments to key steps within the protocol, such as antigen retrieval, can often resolve these common issues.

Problem: Little or No Staining

Sample storage
Slides may lose signal over time in storage. This process is variable and dependent upon the protein target. The effect of slide storage on staining has not been established for every protein; therefore, it is best practice that slides are freshly cut before use. If slides must be stored, do so at 4°C. Do not bake slides before storage.
Tissue sections dried out
It is vital that the tissue sections remain covered in liquid throughout the staining procedure.
Slide preparation
Inadequate deparaffinization may cause spotty, uneven background staining. Repeat the experiment with new sections using fresh xylene.
Antigen unmasking/retrieval
Fixed tissue sections have chemical crosslinks between proteins that, dependent on the tissue and antigen target, may prevent antibody access or mask antigen targets. Antigen unmasking protocols may utilize a hot water bath, microwave, or pressure cooker. Antigen unmasking protocols utilizing a water bath are not recommended. Antigen unmasking performed with a microwave is preferred, though staining of particular tissues or antigen targets may require the use of a pressure cooker.
Unmasking/retrieval buffer
Staining of particular tissues or antigen targets may require an optimized unmasking buffer. Refer to product datasheet for antigen unmasking buffer recommendations. Always prepare fresh 1X solutions daily.
Antibody dilution/diluent
Consult CST™ product datasheet for the recommended dilution and diluent. Titration of the antibody may be required if a reagent other than the one recommended is used.
Incubation time
Primary antibody incubation according to a rigorously tested protocol provides consistent, reliable results. CST™ antibodies have been developed and validated for optimal results when incubated overnight at 4°C.
Detection system
Polymer-based detection reagents, such as SignalStain® Boost IHC Detection Reagents (#8114) and (#8125), in conjunction with SignalStain® DAB Substrate Kit (#8059), are more sensitive than avidin/biotin-based detection systems. Standard secondary antibodies directly conjugated with HRP may not provide sufficient signal amplification. Always verify the expiration date of the detection reagent prior to use.
Negative staining

A complete lack of staining may indicate an issue with the antibody or protocol. Employ a high expressing positive control, such as paraffin-embedded cell pellets, to ensure that the antibody and procedure are working as expected.

Phospho-specific antibodies in particular, or any antibody directed against a rarely expressed protein, may not stain 100% of the cases of a given indication. It is possible that the sample is truly negative.

Problem: High Background

Slide preparation
Inadequate deparaffinization may cause spotty, uneven background staining. Repeat the experiment with new sections using fresh xylene.
Peroxidase quenching
Endogenous peroxidase activity in samples may produce excess background signal if an HRP-based detection system is being used. Quench slides in a 3% H2O2 solution, diluted in RODI water, for 10 min prior to incubation with the primary antibody.
Biotin block
Using biotin-based detection systems with samples that have high levels of endogenous biotin, such as kidney and liver tissues, may be problematic. In this case, use a polymer-based detection system such as SignalStain® Boost IHC Detection Reagents (#8114) and (#8125). A biotin block may also be performed after the normal blocking procedure prior to incubation in primary antibody.
Blocking
Block slides with 1X TBS (#9997) with 5% Normal Goat Serum (#5425) for 30 min prior to incubation with the primary antibody.
Antibody dilution/diluent
Consult CST™ product datasheet for the recommended dilution and diluent. Titration of the antibody may be required if a reagent other than the one recommended is used.
Secondary cross reactivity
The secondary antibody may bind endogenous IgG, causing high background, in some samples where the secondary antibody is raised in the same species as the sample being tested (mouse-on-mouse staining). Include a control slide stained without the primary antibody to confirm whether the secondary antibody is the source of the background.
Washes
Adequate washing is critical for contrasting low background and high signal. Wash slides three times for 5 min with TBST (#9997) after primary and secondary antibody incubations.

posted September 2013

revised November 2013

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