Nacalai USA, Inc. | Product | Simple Staining Procedure: Histofine® One-Step Polymer Detection System

Simple Staining Procedure: Histofine® One-Step Polymer Detection System

Immunohistochemistry

Features

Polymer technology

Multiple molecules of enzyme and secondary antibodies conjugate to amino-acid polymers.
Polymeric enzyme provides increased staining intensity.

Simplified staining steps

Histofine Simple Stain, Conventional Streptabidin-biotin System

Low background

Only Fab' fragments are used as secondary antibodies. As Fab' fragment doesn't bind to endogenous Fc receptor, the background remains clean.

Histofine Simple Stain is a Non-biotin detection system.

Histofine Simple Stain, Conventional Streptabidin-biotin System

Comparison between Histofine and conventional SAB System.

Principle & Procedure

Application Data

Rabbit anti-Keratin/Cytokeratin antibody
Mouse skin stained with –Histofine^®Simple Stain Mouse MAX PO(R) and DAB chromogen. Note cytoplasmic staining of epithelium cells and sweat gland cells.
Rat anti-Mouse CD45R/B220
Mouse spleen stained with –Histofine^®Simple Stain Mouse MAX PO(Rat) and DAB Chromogen. Note membrane staining of almost all lymphocytes in germinal center and scattered interfollicular lymphocytes.
Mouse anti-PCNA antibody (clone:PC10)
Rat liver stained with –Histofine^® Simple Stain Rat MAX PO(MULTI) and DAB chromogen. Note nuclear staining of liver cells.
Mouse anti-PCNA antibody(clone:PC10)

Mouse colon stained with –Histofine^® MOUSESTAIN KIT and DAB chromogen.
Note nuclear staining of Epithelium cells.

Trouble Shooting

Problem	Possible Cause	Solution
No staining or only weak staining results on the positive control slide and the unknown specimen slide.	Drying-out of specimens during staining prior to addition of the reagents.	Never allow the tissue to dry out.
	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.
	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.
	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.
The unknown specimen slide is not stained while the positive control slide is stained.	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.
	Antigen is decomposed by autolysis.	Use tissues obtained by biopsy or surgery, whenever possible.
The backgrounds are intensively stained in all the slides.	[Peroxidase staining]
	Endogenous enzyme activity was not completely blocked.	Ensure the treatment with 3% of hydrogen peroxide added methanol to inactivate endogenous peroxidase activity.
	Non-specific staining is found.	Before adding primary antibody, treat with 10% normal goat or rabbit serum as follows.
		Product	Serum
		Simple Stain™ MAX PO (M)	Goat
		Simple Stain™ MAX PO (R)	Goat
		Simple Stain™ MAX PO (MULTI)	Goat
		Simple Stain™ MAX PO (G)	Rabbit
		Simple Stain™ Mouse MAX PO (R)	Goat
		Simple Stain™ Mouse MAX PO (Rat)	Rabbit
		Simple Stain™ Rat MAX PO (M)	Goat
		Simple Stain™ Rat MAX PO (R)	Goat
		Simple Stain™ Rat MAX PO (G)	Rabbit
		Simple Stain™ Rat MAX PO (MULTI)	Goat
	[Alkaline phosphatase staining]
	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.
	Non-specific staining is found	Before adding primary antibody, treat with 10% normal goat serum.
		Product name	Serum
		Simple Stain™ AP (M)	Goat
		Simple Stain™ AP (R)	Goat
		Simple Stain™ AP (MULTI)	Goat
	Autolysis results in excessive antigens isolated in histological solutions.	Obtain fresh tissues whenever available.
	Insufficient removal of paraffin.	Change xylene or ethanol as the case may be.
	Insufficient washing of antibody.	Ensure thorough washing of antibody.
	A high room temperature accelerates enzyme reactions.	Keep room temperature at 15 to 25°C. Shorten reaction time of enzyme.
	Drying-out of specimens during staining after addition of the reagents.	Never allow the tissue to dry out.
During the reaction, tissue sections come off from the slides.	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.

Technical Report

Technical Report 1 : Application of - Histofine® MOUSESTAIN, Mouse MAX and Rat MAX for mouse and rat frozen tissue sections

Fig. 1 Background comparison between the mouse tissue staining kit and the human tissue staining kit

Fig. 2 Background comparison between the rat tissue staining kit and the human tissue staining kit

Downloads

Immunohistochemistry / Immunocytochemistry (Booklet)

Ordering Information

Enzyme Substrate Systems

Product	Storage	Cat.No.	PKG Size	Price
Histofine DAB-3S Kit (for Peroxidase)	4°C	415192F	500 tests	290.00	Buy
Histofine DAB-3S Kit (for Peroxidase)	4°C	415194F	1500 tests	330.00	Buy
Histofine DAB-2V Kit (for Peroxidase)	4°C	425312F	500 tests	210.00	Buy
Histofine DAB-2V Kit (for Peroxidase)	4°C	425314F	1500 tests	260.00	Buy
Histofine Simple Stain AEC Solution (for Peroxidase)	4°C	415182F	500 tests	300.00	Buy
Histofine Simple Stain AEC Solution (for Peroxidase)	4°C	415184F	1500 tests	370.00	Buy
Histofine New Fuchsin Substrate Kit (for Alkaline phosphatase)	4°C	415161F	2000 tests	210.00	Buy

For Human Tissue Sections

Product	Storage	Cat.No.	PKG Size	Price
Histofine Simple Stain Human MAX PO (MULTI) (for Mouse & Rabbit primary antibody)	4°C	414151F	170 tests	240.00	Buy
Histofine Simple Stain Human MAX PO (MULTI) (for Mouse & Rabbit primary antibody)	4°C	414152F	500 tests	410.00	Buy
Histofine Simple Stain Human MAX PO (MULTI) (for Mouse & Rabbit primary antibody)	4°C	414154F	1500 tests	710.00	Buy
Histofine Simple Stain Human MAX PO (M) (for Mouse primary antibody)	4°C	414131F	170 tests	240.00	Buy
Histofine Simple Stain Human MAX PO (M) (for Mouse primary antibody)	4°C	414132F	500 tests	400.00	Buy
Histofine Simple Stain Human MAX PO (M) (for Mouse primary antibody)	4°C	414134F	1500 tests	680.00	Buy
Histofine Simple Stain Human MAX PO (R) (for Rabbit primary antibody)	4°C	414141F	170 tests	240.00	Buy
Histofine Simple Stain Human MAX PO (R) (for Rabbit primary antibody)	4°C	414142F	500 tests	400.00	Buy
Histofine Simple Stain Human MAX PO (R) (for Rabbit primary antibody)	4°C	414144F	1500 tests	680.00	Buy
Histofine Simple Stain Human MAX PO (G) (for Goat primary antibody)	4°C	414161F	170 tests	260.00	Buy
Histofine Simple Stain Human MAX PO (G) (for Goat primary antibody)	4°C	414162F	500 tests	390.00	Buy
Histofine Simple Stain Human AP (MULTI) (for Mouse & Rabbit primary antibody)	4°C	414261F	170 tests	300.00	Buy
Histofine Simple Stain Human AP (MULTI) (for Mouse & Rabbit primary antibody)	4°C	414262F	500 tests	640.00	Buy
Histofine Simple Stain Human AP (M) (for Mouse primary antibody)	4°C	414241F	170 tests	270.00	Buy
Histofine Simple Stain Human AP (M) (for Mouse primary antibody)	4°C	414242F	500 tests	630.00	Buy
Histofine Simple Stain Human AP (R) (for Rabbit primary antibody)	4°C	414251F	170 tests	270.00	Buy
Histofine Simple Stain Human AP (R) (for Rabbit primary antibody)	4°C	414252F	500 tests	630.00	Buy

For Mouse Tissue Sections

Product	Storage	Cat.No.	PKG Size	Price
Histofine Simple Stain MOUSESTAIN KIT	4°C	414321F	50 tests	400.00	Buy
Histofine Simple Stain MOUSESTAIN KIT	4°C	414322F	500 tests	700.00	Buy
Histofine Simple Stain Mouse MAX PO (R) (for Rabbit primary antibody)	4°C	414341F	170 tests	270.00	Buy
Histofine Simple Stain Mouse MAX PO (G) (for Goat primary antibody)	4°C	414351F	170 tests	270.00	Buy
Histofine Simple Stain Mouse MAX PO (Rat) (for Rat primary antibody)	4°C	414311F	170 tests	270.00	Buy

For Rat Tissue Sections

Product	Storage	Cat.No.	PKG Size	Price
Histofine Simple Stain Rat MAX PO (MULTI) (for Mouse & Rabbit primary antibody)	4°C	414191F	170 tests	270.00	Buy
Histofine Simple Stain Rat MAX PO (M) (for Mouse primary antibody)	4°C	414171F	170 tests	270.00	Buy
Histofine Simple Stain Rat MAX PO (R) (for Rabbit primary antibody)	4°C	414181F	170 tests	270.00	Buy
Histofine Simple Stain Rat MAX PO (G) (for Goat primary antibody)	4°C	414331F	170 tests	270.00	Buy