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N-Histofine® Trouble Shooting

   Go to immunohistochemistry Polymer Detection System top

Trouble Shooting

problem

No staining or only weak staining results on the positive control slide and the unknown specimen slide.

Possible Case
Drying-out of specimens during staining prior to addition of the reagents.
Solution
Never allow the tissue to dry out.

Possible Case
The embedding agent is not suitable, or paraffin is not thoroughly removed from paraffin-embedded sections.
Solution
-Select a suitable embedding agent or remove paraffin thoroughly from sections embedded.
-Change xylene or ethanol as the case may be.

Possible Case
Any trace amount of sodium azide present in the buffer inactivates the peroxidase, making the staining impossible.
Solution
-Use sodium azide free buffer solution.
-Change buffer solution.

Possible Case
Inadequate incubation of the enzyme and antibody.
Solution
-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.

problem

The unknown specimen slide is not stained while the positive control slide is stained.

Possible Case
Antigen is denatured or masked during fixing or embedding process.
Solution
-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.

Possible Case
Antigen is decomposed by autolysis.
Solution
-Use tissues obtained by biopsy or surgery, whenever possible.

problem

The backgrounds are intensively stained in all the slides.

[ Peroxidase staining ]

Possible Case
Endogenous enzyme activity was not completely blocked.
Solution
Ensure the treatment with 3% of hydrogen peroxide added methanol to inactivate endogenous peroxidase activity.

Possible Case
Non-specific staining is found.
Solution
Before adding primary antibody, treat with 10% normal goat or rabbit serum as follows.
Product name Serum
Simple Stain MAX PO (M)
Simple Stain MAX PO (R)
Simple Stain MAX PO (MULTI)
goat
goat
goat
Simple Stain MAX PO (G) rabbit
Simple Stain Mouse MAX PO (Rat) goat
Simple Stain Rat MAX PO (M)
Simple Stain Rat MAX PO (R)
Simple Stain Rat MAX PO (MULTI)
goat
goat
goat

[ Alkaline phosphatase staining ]

Possible Case
Endogenous enzyme activity was not completely blocked.
Solution
Add Levamisole to chromogen/substrate solution. To reduce endogenous enzyme activity, chromogen/substrate solution containing 1mM Levamisole is used.

Possible Case
Non-specific staining is found
Solution
Before adding primary antibody, treat with 10% normal goat serum.
Product name Serum
Simple Stain AP (M)
Simple Stain AP (R)
Simple Stain AP (MULTI)
goat
goat
goat

Possible Case
Autolysis results in excessive antigens isolated in histological solutions.
Solution
Obtain fresh tissues whenever available.

Possible Case
Insufficient removal of paraffin.
Solution
Change xylene or ethanol as the case may be.

Possible Case
Insufficient washing of antibody.
Solution
Ensure thorough washing of antibody.

Possible Case
A high room temperature accelerates enzyme reactions.
Solution
-Keep room temperature at 15 to 25°C.
-Shorten reaction time of enzyme.

Possible Case
Drying-out of specimens during staining after addition of the reagents.
Solution
Never allow the tissue to dry out.

problem

During the reaction, tissue sections come off from the slides.

Possible Case
Some antigens require heat induced antigen retrieval procedure or prolonged reaction time with primary antibody, which may render the sections easily come off.
Solution
Mount tissue sections on slides coated with an adhesive such as 0.02% poly-L-lysine or silane.

NICHIREI Biosciences INC.