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

Immunohistochemistry

-Histofine® Simple Stain MAX PO is a detection reagent designed specifically to allow immunohistochemical staining on formalin-fixed paraffin-embedded human tissue sections. It is the labeled polymer prepared by combining amino acid polymers with peroxidase (PO) and secondary antibody which is reduced to Fab' fragment. To eliminate background staining, solid-phase absorption of secondary antibody is carried out with human serum.

 

Selection Guide

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.
     

Technical Report

Technical Report 1 : IHC Triple-staining Method.

Technical Report 2 : Recommendation of Immunohistochemical polymer detection systems designed for mouse & rat tissue sections. 

(These links above will direct you to Nichirei Biosciences site)

 

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

 

Trouble Shooting

ProblemPossible CauseSolution
  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.
  1. 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.
  1. 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.
  1. 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.
  1. 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.
  1. Antigen is decomposed by autolysis.
  • Use tissues obtained by biopsy or surgery, whenever possible.
  1. 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.
  1. 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]
  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.
  1. 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
  1. Autolysis results in excessive antigens isolated in histological solutions.
  • Obtain fresh tissues whenever available.
  1. Insufficient removal of paraffin.
  • Change xylene or ethanol as the case may be.
  1. Insufficient washing of antibody.
  • Ensure thorough washing of antibody.
  1. A high room temperature accelerates enzyme reactions.
  • Keep room temperature at 15 to 25°C.
  • Shorten reaction time of enzyme.
  1. Drying-out of specimens during staining after addition of the reagents.
  • Never allow the tissue to dry out.
  1. 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.

Downloads

Immunohistochemistry / Immunocytochemistry (Booklet)

Ordering Information

Enzyme Substrate Systems
Product Cat.No. Storage PKG Size Price(US$)  
Histofine DAB-3S Kit (for Peroxidase) 415192F 4°C 500 tests 290.00 Buy
Histofine DAB-3S Kit (for Peroxidase) 415194F 4°C 1500 tests 330.00 Buy
Histofine DAB-2V Kit (for Peroxidase) 425312F 4°C 500 tests 210.00 Buy
Histofine DAB-2V Kit (for Peroxidase) 425314F 4°C 1500 tests 260.00 Buy
Histofine Simple Stain AEC Solution (for Peroxidase) 415182F 4°C 500 tests 300.00 Buy
Histofine Simple Stain AEC Solution (for Peroxidase) 415184F 4°C 1500 tests 370.00 Buy
Histofine New Fuchsin Substrate Kit (for Alkaline phosphatase) 415161F 4°C 2000 tests 210.00 Buy
For Human Tissue Sections
Product Cat.No. Storage PKG Size Price(US$)  
Histofine High Stain Human HRP (MULTI) -High Sensitive 2 Step Polymer Detection System 414481F 4°C 170 tests 640.00 Buy
Histofine High Stain Human HRP (MULTI) -High Sensitive 2 Step Polymer Detection System 414483F 4°C 1000 tests 1,430.00 Buy
Histofine Simple Stain Human MAX PO (MULTI) (for Mouse & Rabbit primary antibody) 414151F 4°C 170 tests 240.00 Buy
Histofine Simple Stain Human MAX PO (MULTI) (for Mouse & Rabbit primary antibody) 414152F 4°C 500 tests 410.00 Buy
Histofine Simple Stain Human MAX PO (MULTI) (for Mouse & Rabbit primary antibody) 414154F 4°C 1500 tests 710.00 Buy
Histofine Simple Stain Human MAX PO (M) (for Mouse primary antibody) 414131F 4°C 170 tests 240.00 Buy
Histofine Simple Stain Human MAX PO (M) (for Mouse primary antibody) 414132F 4°C 500 tests 400.00 Buy
Histofine Simple Stain Human MAX PO (M) (for Mouse primary antibody) 414134F 4°C 1500 tests 680.00 Buy
Histofine Simple Stain Human MAX PO (R) (for Rabbit primary antibody) 414141F 4°C 170 tests 240.00 Buy
Histofine Simple Stain Human MAX PO (R) (for Rabbit primary antibody) 414142F 4°C 500 tests 400.00 Buy
Histofine Simple Stain Human MAX PO (R) (for Rabbit primary antibody) 414144F 4°C 1500 tests 680.00 Buy
Histofine Simple Stain Human MAX PO (G) (for Goat primary antibody) 414161F 4°C 170 tests 260.00 Buy
Histofine Simple Stain Human MAX PO (G) (for Goat primary antibody) 414162F 4°C 500 tests 390.00 Buy
Histofine Simple Stain Human AP (MULTI) (for Mouse & Rabbit primary antibody) 414261F 4°C 170 tests 300.00 Buy
Histofine Simple Stain Human AP (MULTI) (for Mouse & Rabbit primary antibody) 414262F 4°C 500 tests 640.00 Buy
Histofine Simple Stain Human AP (M) (for Mouse primary antibody) 414241F 4°C 170 tests 270.00 Buy
Histofine Simple Stain Human AP (M) (for Mouse primary antibody) 414242F 4°C 500 tests 630.00 Buy
Histofine Simple Stain Human AP (R) (for Rabbit primary antibody) 414251F 4°C 170 tests 270.00 Buy
Histofine Simple Stain Human AP (R) (for Rabbit primary antibody) 414252F 4°C 500 tests 630.00 Buy
For Human Tissue Sections (ALK Detection Kit)
Product Cat.No. Storage PKG Size Price(US$)  
Histofine ALK Detection Kit 417071F 4°C 20 tests 860.00 Buy
Histofine ALK Control Slides 417081F 4°C 5 slides 500.00 Buy
For Mouse Tissue Sections
Product Cat.No. Storage PKG Size Price(US$)  
Histofine Simple Stain MOUSESTAIN KIT 414321F 4°C 50 tests 400.00 Buy
Histofine Simple Stain MOUSESTAIN KIT 414322F 4°C 500 tests 700.00 Buy
Histofine Simple Stain Mouse MAX PO (R) (for Rabbit primary antibody) 414341F 4°C 170 tests 270.00 Buy
Histofine Simple Stain Mouse MAX PO (G) (for Goat primary antibody) 414351F 4°C 170 tests 270.00 Buy
Histofine Simple Stain Mouse MAX PO (Rat) (for Rat primary antibody) 414311F 4°C 170 tests 270.00 Buy
For Rat Tissue Sections
Product Cat.No. Storage PKG Size Price(US$)  
Histofine Simple Stain Rat MAX PO (MULTI) (for Mouse & Rabbit primary antibody) 414191F 4°C 170 tests 270.00 Buy
Histofine Simple Stain Rat MAX PO (M) (for Mouse primary antibody) 414171F 4°C 170 tests 270.00 Buy
Histofine Simple Stain Rat MAX PO (R) (for Rabbit primary antibody) 414181F 4°C 170 tests 270.00 Buy
Histofine Simple Stain Rat MAX PO (G) (for Goat primary antibody) 414331F 4°C 170 tests 270.00 Buy

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