While using IHC, there may be some barriers occur. Let’s overcome these by way of IHC Troubleshooting Tips:
PROBLEM 1: A few or no Staining
In the case of a few or no staining, the following solutions may use:
Tissue sections dried out: For this, it is essential that tissue sections remain closed in liquid during the entire staining procedure.
Slide preparation: In the case of Inadequate deparaffinization may result in uneven background staining. For this, repeat the experiment with new sections with the help of fresh xylene.
Retrieval buffer: Make sure to strain particular tissues or antigen targets may need an optimized unmasking buffer. For this, take the help of product datasheet for antigen unmasking buffer recommendations. Moreover, always prepare fresh 1X solutions regularly.
Negative staining: In this case, an entire lack of staining may show an issue with the antibody or protocol. For this, use a high expressing positive control, like paraffin-embedded cell pellets, to provide that the antibody and process are working accordingly.
Sample storage: For this, slides may lose signal over time in storage. The effect of slide storage on staining has not been set for every protein so that it will be the best that slides are freshly cut before use. Moreover, if slides must be stored, then do so at 4°C. Moreover, never bake slides before storage.
Antigen unmasking/retrieval: For Antigen Retrieval Methods, the Antigen unmasking protocols may use a hot water bath or microwave. It may be noted that antigen unmasking protocols utilizing a water bath are not suggested. For this, microwave Antigen unmasking should be performed.
PROBLEM 2: High Background
In the case of the High Background, the following solutions may use:
Peroxidase quenching: Because of endogenous peroxidase activity in samples may result in excess background signal in case of an HRP-based detection system used. For this, break slides in a 3% H2O2 solution and then diluted in RODI water, for 10-15 minutes before incubation with the primary antibody.
Slide preparation: Insufficientdeparaffinization may result in uneven background staining. For this, repeat the experiment with new sections with the help of fresh xylene.
Washes: Sufficient cleaning is required for contrasting low background and high signal. For this, clean slides at least 3-4 times for 5-10 minutes with TBST (#9997) after primary and secondary antibody incubations.
Blocking: In a case of blocking, the block slides with 1X TBST with 5% Normal Goat Serum for half an hour before incubation with the primary antibody.
Biotin block: By using the biotin-based detection systems having high levels of endogenous biotins, like liver and kidney tissues, may be problematic. In this circumstance, make sure to use a polymer-based detection system like SignalStain® Boost IHC Detection Reagents. In addition, a biotin block may also be executed after the normal blocking process before incubation in primary antibody.