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  • Non specific negative control staining
  • No Positive control staining

There are several reasons for why non-specific staining is being seen in the negative control as shown in the table below.

Reasons

Solution

Inadequate or delayed fixation of the tissue

Make sure tissue has been adequately fixed throughout

Endogenous peroxidise activity leading to background staining

Pretreatment of the tissue section with hydrogen peroxide prior to incubation of primary antibody.

Primary antibody same species as tissue tested eg mouse primary antibody for mouse control tissue

Make sure that the primary antibody and the tissue are from different species.

Sections dried out

Make sure the sections are kept well hydrated at all times

Too much amplification

Reduce amplification incubation time or dilute the amp kit

Over retrieval

Carry out testing of retrieval protocols until the procedure can be validated as acceptable for general usage.
No staining in the Positive control As you can see on the list below, there are several reasons for why a positive control would not show the expected staining required.

Problem

Solution

Antibody Expired

Replace with new antibody and test to confirm it is now effective, before putting into general usage.

Staining blocked by resin

Treat resin cases in xylene for around 12 hours before carrying out ICC staining.

Antibody too dilute

Make up a less dilute antibody and test before putting into general use.

Inappropriate fixative eg Ethanol or picric acid

Only perform immunostaining procedure on sections where fixation has been carried out in a suitable fixative.