Print

Home>Technical Information>Trouble Shooting

Trouble Shooting

Problem Possible Case Solution
1.  No staining or only weak staining results on the positive control slide and the unknown specimen slide.
1)  Drying-out of specimens during staining prior to addition of the reagents.
Never allow the tissue to dry out.
2)  The embedding agent is not suitable, or paraffin is not thoroughly removed from paraffin-embedded sections.
Select a suitable embedding agent or remove paraffin thoroughly from sections embedded.
Change xylene or ethanol as the case may be.
3)  Any trace amount of sodium azide present in the buffer inactivates the peroxidase, making the staining impossible.
Use sodium azide free buffer solution.
Change buffer solution.
4)  Inadequate incubation of the enzyme and antibody.
Change stale chromogen/substrate reagent.
Blot off excess solution thoroughly at each stage.
Provide sufficient time for reaction with antibody. In particular, primary antibody should be incubated for the time period specified in the insert.
2.  The unknown specimen slide is not stained while the positive control slide is stained.
1)  Antigen is denatured or masked during fixing or embedding process.
Some antigens are sensitive to fixation or embedding. So use less potent fixative and decrease the fixing time.
The pretreatment is required for some tissues, in order to reveal the antigen, such as Antigen Recovery, Heat-Induced Epitope Retrieval or trypsin treatment.
2)  Antigen is decomposed by autolysis.
Use tissues obtained by biopsy or surgery, whenever possible.
3.  The backgrounds are intensively stained in all the slides.
[ Peroxidase staining ]
1)  Endogenous enzyme activity was not completely blocked.
 
Ensure the treatment with 3% of hydrogen peroxide added methanol to inactivate endogenous peroxidase activity.
2)  Non-specific staining is found.
Before adding primary antibody, treat with 10% normal goat or rabbit serum as follows.
Product name Serum
Simple StainTM MAX PO (M)
Simple StainTM MAX PO (R)
Simple StainTM MAX PO (MULTI)
goat
goat
goat
Simple StainTM MAX PO (G) rabbit
Simple StainTM Mouse MAX PO (R)
Simple StainTM Mouse MAX PO (G)
Simple StainTM Mouse MAX PO (Rat)
goat
rabbit
goat
Simple StainTM Rat MAX PO (M)
Simple StainTM Rat MAX PO (R)
Simple StainTM Rat MAX PO (G)
Simple StainTM Rat MAX PO (MULTI)
goat
goat
rabbit
goat
[ Alkaline phosphatase staining ]
1)  Endogenous enzyme activity was not completely blocked.
 
Add Levamisole to chromogen/substrate solution. To reduce endogenous enzyme activity, chromogen/substrate solution containing 1mM Levamisole is used.
2)  Non-specific staining is found
Before adding primary antibody, treat with 10% normal goat serum.
Product name Serum
Simple StainTM AP (M)
Simple StainTM AP (R)
Simple StainTM AP (MULTI)
goat
goat
goat
3)  Autolysis results in excessive antigens isolated in histological solutions.
Obtain fresh tissues whenever available.
4)  Insufficient removal of paraffin.
Change xylene or ethanol as the case may be.
5)  Insufficient washing of antibody.
Ensure thorough washing of antibody.
6)  A high room temperature accelerates enzyme reactions.
Keep room temperature at 15 to 25C.
Shorten reaction time of enzyme.
7)  Drying-out of specimens during staining after addition of the reagents.
Never allow the tissue to dry out.
4.  During the reaction, tissue sections come off from the slides.
1)  Some antigens require heat induced antigen retrieval procedure or prolonged reaction time with primary antibody, which may render the sections easily come off.
Mount tissue sections on slides coated with an adhesive such as 0.02% poly-L-lysine or silane.
BACK

Page Top