Let’s find out the comprehensive guide on IHC Troubleshooting Overstaining to enhance optimization. Let’s highlight the reasons when there is a need to check Troubleshooting:

High Background
• Cleaning of sections was insufficient: For this, wash at least 3 instances among steps.
• Endogenous enzyme which includes peroxidase or alkaline phosphatase became active: If so, then block endogenous enzyme activity by using 3% hydrogen peroxide in methanol solution previous to incubation of primary antibody.
• Washing of sections was inadequate: If so, wash it at least 4-5 times between steps.
• Mouse antibody was used on mouse tissues: Treat tissue with Mouse on Mouse blocking reagent prior to the primary antibody incubation
• Sections dried out: Avoid sections being dried out.
• Incubation temperature may be too high: Incubate sections or cells at 4°C.
• Overstaining due to over amplification: Reduce amplification incubation time and dilute the secondary antibody.

No Staining
• Antibody turned into inactive due to flawed storage: If so, then aliquot antibodies into tiny volumes and keep in the freezer as well and keep away from repeated freeze and thaw cycles.
• Fixation of tissue changed into too long: In this case, decline the duration of postfixation. In any circumstance, tissue has already been overfixed, optimized antigen retrieval way to unmask the epitope.
• Deparaffinization was incorrect: If Deparaffinize sections become longer, then use fresh xylene solution.
• Antibody become unsuitable for IHC methods: In a case, if it's been validated in IHC, and what form of IHC. Examine the antibody in a negative WB to make certain it isn't damaged.
• Fixation of tissue was inadequate or improper: Increase duration of postfixation or try different fixatives.
• Antibody concentration became insufficient: Hike up the attention or execute a serial dilution check to decide the maximum dilution that offers the great sign to noise ratio. Or incubate longer at 4°C.
• Eliminate system changed in incompatible: Substitute with a distinctive substrate system that's well suited with enzyme.

Overstaining
• Antibody concentration became too high: If so, decline concentration or carry out a titration to determine the most useful dilution for antibody
• Eliminate incubation time became too long: Decline substrate incubation time
• Incubation time turned into too long: If so, decline incubation time
• The incubation temperature turned into too excessive: Decline incubation temperature
• Sections dried out: If so, make sure to avoid sections being dried out

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