SPIROCHETE Treponema sp. Control Slides

Description

SPIROCHETE Treponema sp. Control Slides

These quality control slides are intended to be used to verify histological techniques and reagent reactivity.  They are to be used for the qualitative purpose of determining positive or negative results, and are not intended to be used for any quantitative purpose.  The first serial section within this control box is stained and provided for your reference. 

Newcomer Supply, Inc., takes reasonable measures to deliver quality control slides that are as consistent as possible. However, the characteristics of quality control slides may be dissimilar due to variations in the reagents, stains, techniques, laboratory conditions, and tissue sources used.

Spirochete (Treponema pallidum) is the causative agent of syphilis.  In the past, identification of the spirochete organisms was achieved with silver staining methods.  Treponema pallidum can now be successfully localized with immunohistochemistry techniques.

These Spirochete control slides are produced from animal tissues under carefully controlled conditions.  At Newcomer Supply we use a manual method of performing immunohistochemical stains, specifically avoiding automation, in order to provide reactive control slides for even the less aggressive methods of staining that our customers may be using.

Please note: A section of normal human lung is included on the slide for use as a negative control.

METHOD:

Fixation:                    10% Neutral Buffered Formalin                                                                        

Technique:                Paraffin sections cut at approximately 4 microns

REAGENTS:

Epitope Retrieval:

Target Retrieval Solution, Dako

Catalog #S1699

Diluent:

Antibody Diluent, Cell Marque

Catalog #936B

Blocking Reagent:

3% Hydrogen Peroxide, Newcomer Supply, (30%)

Catalog #1206A

Primary Antibody:

Treponema pallidum (Spirochete) (1:50), Biocare

Catalog #CP 135

Negative Antibody:

Rabbit Negative Control Serum, Cell Marque

Catalog #933B-02

Detection Kit:

HiDef Detection^TM HRP Polymer System, Cell Marque

Catalog #954D-20

Chromogen:

DAB Substrate Kit, Cell Marque

Catalog #957D-10

Buffer:

Tris Buffered Saline, 0.05M, pH 7.6, 10X, Newcomer Supply

Catalog #140304

Hematoxylin:

Hematoxylin Stain Solution, Mayer Modified, Newcomer Supply

Catalog #1202

STAINING PROCEDURE - Manual Method:

1. Deparaffinize sections thoroughly in 3 changes of xylene, then hydrate through graded ethyl alcohols to distilled water.
2. Place slides in preheated (95^oC) Target Retrieval Solution in steamer for 20 minutes.  Remove and allow to cool for 20 minutes at room temperature.
3. Rinse in 3 changes of buffer ending with a distilled water rinse.
4. Tap off excess water and apply enough freshly made 3% Hydrogen Peroxide to cover specimen. Incubate for 5 minutes.
5. Rinse slides gently in distilled water from a wash bottle and place in buffer bath.
6. Tap off excess buffer and apply enough primary antibody (1:50) or negative control reagent to cover specimen.  Incubate for 30 minutes.
7. Rinse slides gently in buffer from a wash bottle, then place in buffer bath for 5 minutes.
8. Tap off excess buffer and apply enough Amplifier to cover specimen.  Incubate for 10 minutes.
9. Rinse slides gently in buffer from a wash bottle and place in buffer bath.
10. Tap off excess buffer and apply enough HRP Polymer to cover specimen.  Incubate for 10 minutes.
11. Rinse slides gently in buffer from a wash bottle and place in buffer bath.
12. Prepare enough substrate-chromogen DAB for the amount of slides being stained.
13. Tap off excess buffer and apply enough DAB to cover specimen.  Incubate for 5 minutes.
14. Rinse slides gently in a distilled water from a wash bottle, then place in distilled water bath.
15. Counterstain lightly with Hematoxylin Stain Solution, Mayer Modified or other aqueous hematoxylin.
16. Blue sections in warm tap water.
17. Dehydrate sections through graded ethyl alcohols, clear in xylene or substitute and coverslip with a compatible mounting medium.

RESULTS:

• Spirochete positive expression                              Brown
• Normal Human Lung                                               Negative         

REFERENCES:

1. Cell Marque HiDef Detection^TM Polymer System datasheet.
2. Biocare Treponema pallidumantibody datasheet.