HistoGreen, HRP-Substrate-Kit (enough for 1,000 sections) Result: Green Color

Substrate for Immunohistochemistry

SKU:
E109
Category
Quantity
1 kit

Background

HistoGreen is a very sensitive substrate chromogen for use in peroxidase-based immuno-histochemical (IHC) and in situ-hybridisation (ISH) staining procedures. It is perfectly suitable for single as well as for multi staining. The HistoGreen substrate kit contains all the reagents necessary to prepare the working solution for application. HistoGreen produces with the peroxidase a green reaction product that can be only permanently mounted. It produces with peroxidase and alkaline phosphatase substrates an excellent contrast and is compatible with the counterstains HistoHematoxylin and HistoNuclear Fast Red.
In contrast to DAB HistoGreen is neither toxic nor carcinogen.

The reagents are sufficient for approx. 1000 tissue sections.

  • 1 dropbottle HistoGreenChromogen (bottle No 1, 8 ml)
  • 1 bottle HistoGreen-Buffer (bottle No 2, 100 ml)
  • 1 dropbottle H2O2 (bottle No 3, 8 ml)
  • 1 dropbottle (empty) for the working dilution (G)

For convenience the reagents are supplied in dropper bottles. When dispensing drops, hold the bottle in an inverted vertical position and squeeze gently. To prevent evaporation, secure the opaque caps on the bottle when they are not in use. DO NOT PIPET REAGENTS DIRECTLY FROM THE BOTTLE. Proper concentrations of substrate components are assured in preparing the working solution by using the drop dispenser.

Prepare the HistoGreen substrate solution directly before used as follows:

Ues empty bottle G for preparation of working substrate solution:

  • add 1 ml HistoGreen-Buffer from (bottle No 2) to empty bottle G
  • add 2 drops from dropbottle 1 HistoGreenChromogen (bottle No 1)
  • add to this mixture 2 drops H2O2 (bottleNo 3) and mix well.

Rinse the sections before adding the substrate in TBS-buffer (0.05 M TRIS-Base, 0.15 M NaCl, pH 7.2) or PBS by Dulbecco (DPBS) (LINARIS CatNr. GBP1622AL powder, GBF1622YK liquid). Incubate the sections with the substrate solution at room temperature while watching until a sufficient colour intensity is reached. Generally a good staining result should be reached within 1-5 minutes. Wash the stained sections for 2-5 min in TBS or DPBS-buffer and rinse them shortly in aqua. dest.

If prefered, the stained section may be counterstained with HistoHematoxylin (LINARIS CatNr EGH3401) or HistoNuclearFast Red (LINARIS CatNr EGH3403).

The reaction product of the HistoGreen-Substrate is water-soluble and has to be dehydrated in series of alcohol before mounting and has to be mounted with VectamountTM (LINARIS CatNr E6003) or similar mounting mediums.

Recommendation for Dehydration

The substrate may fade when kept long in the single alcohol components of the dehydration series.
We recommend only 30 sec. per component (better: rinse carefully) and the following dehydration series:
Alcohol pure 100% – alcohol pure 100% – alcohol purest 100% – Xylene pure – Xylene pure

Additional Notes

1. HistoGreen should be stored at 4°C and be kept dark. The colour of the staining substrate may get a little dark in course of time without affecting the staining quality of HistoGreen.

2. Do not heat up single components or working dilution of the substrate, max. 25°C.

3. Use only Xylene of the „pure“ quality and do not exceed 30 sec. for every cuvette!

4. Do not mount HistoGreen stained sections in aqueous mounting medias!

5. Extended incubation times in TBS, DPBS, alcohol or Xylene may reduce the colour intensity.

6. When used in double or multiple staining in combination with other substrates for Peroxidase, HistoGreen should be used as the last substrate component for the best results.

Trouble Shooting

The substrate may generate a dark blue reaction product in some cases:

  • Impure Xylene or Xylene substitutes are used in the dehydration of the stained sections.
  • Use only Xylene of the „pure“ quality
  • Substrate reaction is too intensive (high expressed antigens):
  • Dilute the primary antibody and/or the HRP-conjugate strongly and shorten the incubation time of the substrate solution.
  • Substrate was heated over 25°C.
  • Use at room temperature only.

Substrate fades or merges:

  • Substrate waSections weren’t dehydrated and aqueous mounting medias were used:
  • Dehydrate stained sections and use only non-aqueous mounting medias.
  • Chemical reactions as X-Gal-development were done after the development of HistoGreen:
  • Use and develop HistoGreen always as last the component.
  • The keeping in the single components of the dehydration was too long
  • Use and develop HistoGreen always as last the component.
  • U. Kämmerer et al. (2003). Expression of Tumor Markers on Breast and Ovarian Cancer Cell Lines. Anticancer Research 23:1051-1056
  • U. Kämmerer et al. (2003). Immunocompetent cells in the endometrium of fetuses and children. Human Reproduction 18(5):969-975
  • U. Kämmerer et al. (2003). Unique Appearance of Proliferating Antigen-Presenting Cells Expressing DC-SIGN (CD209) in the Decidua of early Human Pregnancy. American Journal of Pathology 162(3):887-896
  • Thomas MA, Lemmer B. (2005). HistoGreen: A new alternative to 3,3′-diaminobenzidine-tetrahydrochloride-dihydrate (DAB) as a peroxidase substrate in immunohistochemistry? Brain Res Brain Res Protoc. 2005 Feb;14 (2):107-18.

Application

IHC-F, IHC-P, ISH

Quantity

1 kit

Storage Temperature

2-8 °C

Shipping Temperature

20 °C

Documents

pdf Datasheet