Paraffin section anti-tartaric acid phosphatase (TRAP) immunohistochemical staining kit instructions

January 17 11:09:18, 2024

Paraffin section anti-tartaric acid phosphatase (TRAP) immunohistochemical staining kit
The main purpose
Paraffin section anti-tartaric acid phosphatase (TRAP) immunohistochemical staining reagent is a technique for the use of standardized chemical separation paraffin method and immunological antibody chromogenic technology, namely by anti-tartaric acid phosphatase polyclonal antibody and horseradish peroxide The enzyme conjugate secondary antibody, stained with dye diaminobenzidine, showed a tan, an authoritative and classical technique for analyzing the expression and distribution of tartaric acid phosphatase in paraffin-embedded tissue sections in archives. The technology has been carefully developed and successfully tested. It is mainly applied to the analysis of the expression of tartrate-resistant acid phosphatase in various human, rat, mouse and other osteoclast tissues embedded in paraffin. It can be used for the identification of studies on the pathophysiology of bone cells. The product is strictly sterile, ready to use, clear color, simple operation and stable performance.

technical background
Tartrate resistant acid phosphatase (TRAP) is a glycosylated monomeric metalloproteinase expressed in mammals, 970 polybases, more than 320 amino acids, and a molecular weight of 35 kD. As a marker of osteoclasts, tartrate-resistant acid phosphatase is involved in bone resorption activity and osteogenic metabolism regulated by osteoclasts. The tartrate-resistant acid phosphatase is synthesized in the form of a zymogen, activated by protease cleavage and reduction. In an acidic environment, the hydrolysis of phosphate linkages is catalyzed to produce alcohol and release phosphoric acid. It is characterized by tartaric acid resistance and is distinguished from other acid phosphatases. Tartrate-resistant acid phosphatase is highly expressed in osteoclasts, activated macrophages, nerve cells, and porcine endothelial endometrium in pregnancy. In leukaemic reticuloendotheliosis or hairy cell leukaemia, Gaucher's disease, HIV-induced encephalopathy, osteocytoma, osteoid The anti-tartaric acid phosphatase is significantly increased in patients with osteoporosis and metabolic bone disease. The anti-tartaric acid acid phosphatase expression-positive osteoclast was tanned by a polyclonal antibody against tartrate acid phosphatase and a Peroxidase conjugated antibody, followed by dye diaminobenzidine staining. .

product content
De350 (Reagent A) 300 ml (bring your own)
Reagent B 100 ml hydrating solution B (Reagent C) 100 ml hydrating liquid C (Reagent D) 100 ml hydrating liquid D (Reagent E) 100 ml cleaning solution (Reagent F) 120 ml repair solution (Reagent G) 2 ml blocking solution (Reagent H) 4 ml primary solution (Reagent I) 1 ml secondary solution (Reagent J) 1 ml of coloring solution A (Reagent K) 500 μl of color developing solution B (Reagent L) 5 ml 1 product manual
storage method
Reagent G, Reagent I, Reagent J and Reagent K in the -20 ° C refrigerator, strictly avoid repeated freezing and thawing, the rest are stored in 4 °C refrigerator; second liquid (Reagent J) and coloring liquid A (Reagent K), avoiding light; effective guarantee for March
User-supplied
Small Dyeing Cylinder: Dewaxing and Dyeing for Paraffin Sections Cover Slide: For Antibody Incubation Operation Light Microscopy: For Cell Analysis and Analysis
pay attention
  • Coloring solution A (Reagent K ) and coloring solution B (Reagent L ) must be mixed immediately before the experimental operation; after mixing, avoid using it for a long time or storing it for later use; throw away
  • If the precipitation solution A (Reagent K ) is precipitated, it must be filtered before use.

Experimental procedure
  • Dewaxing
  • Remove 10 pieces of 4 to 10 micron thick paraffin-embedded tissue sections (note: before paraffin embedding, tissue sections must be fixed with 10 % formaldehyde at 4 °C for 16 hours. This step is important )
  • (Selection step) Put in 80 ° C oven, incubate for 30 minutes
  • (Selection step) Let stand for 15 minutes at room temperature
  • Into the small staining tank and incubate according to the following table
Dyeing cylinder Incubation time
50 ml dewaxing liquid ( Reagent A ) 15 minutes
50 ml dewaxing liquid ( Reagent A ) 15 minutes
50 ml dewaxing liquid ( Reagent A ) 15 minutes
50 ml hydrating solution A ( Reagent B ) 3 minutes
50 ml hydrating liquid B ( Reagent C ) 3 minutes
50 ml hydrating liquid C ( Reagent D ) 3 minutes
50 ml hydrating liquid D ( Reagent E ) 3 minutes
  • Carefully remove the hydrating fluid D ( Reagent E ) from the slice
  • Carefully add 200 μl of cleaning solution ( Reagent F ) on the slice to cover the entire surface of the sample.
  • Incubate for 5 minutes at room temperature
Carefully remove the cleaning solution from the slice ( Reagent F )
Second, the sample repair process
  • Carefully add 200 μl of Repair Solution (Reagent G ) to cover the entire surface of the sample.
  • Incubate for 5 minutes at room temperature
  • Carefully remove the repair solution on the slice (Reagent G )
  • Incubate the sections in 50 ml of clear solution (Reagent F ) for 5 minutes at room temperature.
  • Carefully remove the cleaning solution from the slice (Reagent F )
  • Carefully add 200 μl of blocking solution (Reagent H ) to cover the entire surface of the sample.
  • Incubate for 30 minutes at room temperature
  • Carefully remove the blocking solution on the slice (Reagent H )
  • Incubate the sections in 50 ml of clear solution (Reagent F ) for 5 minutes at room temperature.
  • Carefully remove the cleaning solution from the slice (Reagent F )
  • Immune labeling process
  • Carefully add 100 μl of a solution (Reagent I ) to cover the entire surface of the sample.
  • Incubate for 1 hour at room temperature, keep moist and avoid drying (Note: you can use a cover slip to incubate)
  • Carefully remove the primary antibody on the slice (Reagent I )
  • Carefully incubate the sections in 50 ml of clear solution (Reagent F ) for 2 minutes at room temperature.
  • Carefully remove the cleaning solution from the slice (Reagent F )

Fourth, sample dyeing treatment
Before dyeing, the color developing solution A (Reagent K) -20 ℃ refrigerator kit slot into the ice melted, then pipette 200 [mu] l of color reagent A (Reagent K) color developing solution and 1.8 ml B (Reagent L ) into a 2 ml centrifuge tube, mix well, mark as a color developing solution , and place in a dark room. Then do the following:
  • Carefully add 100 μl of secondary antibody (Reagent J ) on the section and spread the entire surface of the sample (note: you can use the coverslip to incubate)
  • Incubate for 30 minutes at room temperature to avoid light
  • Carefully remove the secondary antibody on the slice (Reagent J )
  • Carefully incubate the sections in 50 ml of clear solution (Reagent F ) for 2 minutes at room temperature.
  • Carefully remove the cleaning solution from the slice (Reagent F )
  • Carefully add 200 microliters containing color developing solution A (Reagent K) and color reagent B (Reagent L) color working fluid covered the entire surface of the slice sample
  • Incubate for 5 to 60 minutes at room temperature or until the sample appears tan
  • Carefully remove the slices containing color developing solution A (Reagent K) and color reagent B (Reagent L) color fluid
  • At room temperature, carefully place the slices in 50 ml of clean solution (Reagent F ) for 5 seconds.
  • Carefully remove the cleaning solution from the slice (Reagent F )
  • (Selection step) for methyl green or hematoxylin counterstaining (Note: methyl green counterstaining kit is recommended - YJ 40010 )
  • Put a cover slip or cover
  • Immediate observation under a general light microscope: positive cells appear tan

Precautions
  • This product is 10 operations
  • Wear gloves to prevent contamination during operation
  • Desolvent (Reagent A) can be replaced with xylene
  • Cleaning liquid (Reagent F) 50 mL placed in a small tank dyeing, reusable
  • All operations are performed at room temperature
  • Keep the sliced ​​surface almost dry each time the reagent solution is changed. Dry in the air, no water droplets, wet state, visible reflection
  • When the reagent solution is slicing the surface, avoid the presence of air bubbles while ensuring that the surface of the slice is covered.
  • Cell color development, avoid excessive, close visual observation, control time
  • Immediately after observation of the color of the cells, optical microscopy
  • The company provides a series of osteoclast detection reagent products

Quality Standard
  • This product has been certified to be stable.
  • This product has been identified and clearly colored

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