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Catalog

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Brochure

Simple Stain™ MAX PO

High Stain™ HRP (MULTI)

For Mouse on Mouse

For Mouse and Rat Tissue Sections

For ALK Detection Kit

ALK Control Slides

DAB-2V

Application Guide

Technical Information

● IHC Triple-staining Method

● Recommendation of Immunohistochemical polymer detection system designed for mouse & rat tissue sections

● Advantage of N-Histofine®

● Staining Procedure

Trouble Shooting

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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 Stain^TM MAX PO (M) Simple Stain^TM MAX PO (R) Simple Stain^TM MAX PO (MULTI)	goat goat goat
Simple Stain^TM MAX PO (G)	rabbit
Simple Stain^TM Mouse MAX PO (R) Simple Stain^TM Mouse MAX PO (G) Simple Stain^TM Mouse MAX PO (Rat)	goat rabbit goat
Simple Stain^TM Rat MAX PO (M) Simple Stain^TM Rat MAX PO (R) Simple Stain^TM Rat MAX PO (G) Simple Stain^TM 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 Stain^TM AP (M) Simple Stain^TM AP (R) Simple Stain^TM 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 25°C.
–	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.

Staining is not observed or weak staining is observed on positive control slide and specimen slide.

■ Possible cause & Solution

Possible cause	Solution
1.Drying-out of tissue sections during staining prior to addition of reagents.	・Prevent tissue sections from drying out.
2.Embedding agent used is not suitable, or paraffin is not thoroughly removed from paraffin-embedded tissue sections.	・Use suitable embedding agent or remove paraffin thoroughly from tissue sections embedded. ・Change xylene or ethanol in some cases.
3.Any trace amount of sodium azide present in buffer inactivates peroxidase, such staining may not be available.	・Use sodium azide free buffer solution. ・Change buffer solution.
4.Inadequate incubation of enzyme and antibody.	・Change stale chromogen-substrate reagent. ・Remove excess solution thoroughly at each stage. ・Allow antibody sufficient time to react. In particular, primary antibody should be incubated for the specified time in its instruction for use.

Backgrounds are intensively stained on all the slides.

■ Possible cause & Solution

Peroxidase staining

Possible cause	Solution
1.Endogenous enzyme activity was not completely blocked.	・Ensure 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.
3.Amount of antigen is few in tissue sections.	・Prolong reaction time of reagent at each step.

Possible cause

Solution

1.Endogenous enzyme activity was not completely blocked.

・Ensure 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.

3.Amount of antigen is few in tissue sections.

・Prolong reaction time of reagent at each step.

Alkaline phosphatase staining

Possible cause	Solution
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 should be used.
2.Non-specific staining is found.	・Before adding primary antibody, treat with 10% normal goat serum as follows.
3.Autolysis results in excessive antigens isolated in histological solutions.	・Use fresh tissues whenever available.
4.Insufficient removal of paraffin.	・AChange xylene or ethanol in some cases.
5.Insufficient washing of antibody.	・Ensure thorough washing of antibody.
6.High room temperature accelerates enzyme reactions.	・Keep room temperature at 15 to 25°C. ・Shorten reaction time of enzyme.
7.Tissue sections are dried out during staining after addition of reagents.	・Prevent tissue sections from drying out.