Tips or guide requires to following to target the main cause of a barrier with your protocol and test out outcomes. Or this, it is recommended to identify the problem with your IHC staining from the following options:
High Background
Non-specific Staining
Weak or No Staining
IN CASE HIGH BACKGROUND: Make sure to consider the following IHC Troubleshooting Tips if you are tackling high background.
If Deparaffinization is not abundant: Make sure to indulge in fresh xylene and keep the Deparaffinize sections longer.
In a case Antibody Concentration is too up: Then Dilute the primary and/or secondary antibody more.
If blocking inadequate: Then hike up the blocking incubation period as well as considering to amending the blocking agent.
If inadequate washing: Then wash it properly. Make sure to follow the protocol guidelines while indulging in washing steps.
IN A CASE OF NON-SPECIFIC STAINING: Make sure to consider the following tips if you are tackling non-specific staining.
If Antibody Concentrations are too up: Make sure to decline the concentration as well as the incubation period.
IN CASE OF WEAK OR NO STAINING: Make sure to consider the following tips if you are tackling weak or no staining.
In case of not sufficient primary antibody: Make sure to use a higher concentration of antibody
If primary and secondary antibodies are opposed: In this case, the secondary antibody should be upgraded against the host of the primary antibody. Moreover, Isotypes must be compatible as well.
If Deparaffinization is not enough: In this circumstance, make sure to use fresh xylene as well Deparaffinize sections should be longer.
If the tissue is dried out: Then samples should be kept covered in liquid during the staining process.
If cells were not permeabilized: Then, use Methanol and acetone fixation to permeabilize cells
If tissues are over constant: Then, decline the duration of fixation as well as indulge in using the various antigen retrieval methods to unmask the epitope.
If Incubation time is too small: Then hike up the duration of incubation of the primary antibody with the sample
In the case of Slide storage issues: Then samples must be imaged quickly after processing as the signal declines over time.
If Antibody Storage issues: Then no need to store Antibody as recommended. For this, there will be a need for the new vial to be used.
If protein is not available in the tissues being tested: In this, execute a positive control. Moreover, in a circumstance, if the protein is available but not sufficient, then use an amplification process to hike up the signal.
Booster Antibody and Elisa experts is a prominent destination to get more guidance about the IHC Troubleshooting problems. It has veteran experts that can guide you thoroughly about the IHC Troubleshooting Tips until you become satisfied. Paraffin Embedding involves the steps fixation, dehydration, transparentizing, immersion and embedding. Paraffin Embedded Tissue is sliced by a rotary microtome to give a thickness of 2-7 µm.
