TROUBLESHOOTING

Question 1

Both specimen and positive control are unstained

Answer 1

1. Primary antibody was not applied to tissue
2. Reagents were applied in the wrong order or the incorrect counterstain was used with AEC

3.Wrong secondary antibody was used

4. Tissue sections were allowed to dry during staining

Question 2

Specimen unstained, positive control stained

Answer 2

1.Substrate- chromogen was improperly prepared

2. Primary Ab too dilute or not properly prepared
3. Insufficient incubation time
4. To much buffer was left on the slides during staining
5. The Epitope enhancement was incorrectly done

Question 3

Specimen has weak staining but the positive control stained

Answer 3

1. The paraffin was incompletely removed
2. Too many cells contain endogenous peroxidase and haven’t been adequately “quenched”
3. Excessive adhesive was used
4. The slides were not washed well with reaction buffer
5. Incubation time of either the primary or secondary antibody was too long or the labeled reagent was too high

Question 4

Excessive background staining in the patient tissue but none in the control

Answer 4

Free antigen is present in tissue due to: necrosis, autolysis or degeneration

Question 5

One area within specimen shows
appropriate ‘reactivity’, while other areas do not, may appear as a “tie dyed” effect

Answer 5

Incomplete deparaffinization, slides were not placed onto the machine level or there was an inconsistent reagent volume during processing

Question 6

False Negative

Answer 6

Antigen loss
Inadequate ag retrieval
Method not sensitive enough
Expired ab
Wrong reagents or wrong reagent sequence