Animal glycoprotein Hill periodate method Alcian blue staining kit instructions product (Chinese)
The main purpose
Animal Cell Glycoprotein Periodic Acid Hilfa Alcian Blue Staining Reagent is an authoritative method for the complete and complementary analysis of proteoglycan (glycoprotein) components in cell samples by a combination of Alcian blue and periodic acid Hill methods. And the classic technical method. The technology has been proven through careful improvement of traditional methods and successful experiments. It is mainly used for glycoprotein components in various animal culture cells, especially for the detection of acidic and neutral mucopolysaccharide proteins. The product is ready to use, simple operation, stable performance and clear color.
technical background
The alcian blue dye is a basic dye of the water-soluble copper phthalocyanine group. It appears blue due to the presence of copper in the molecule. In a low pH environment, the reaction of arsenic blue with polyvalent anionic sulfated and carboxylated free radicals, such as acid mucopolysaccharide, salam mucopolysaccharide (sialomucins) And a glycoprotein, which forms a salt linkage with an acidic group, exhibiting a blue color (usually periodicity of the periodic acid). Periodic acid oxidizes polysaccharide glycosyl groups in glycoproteins, including carbon atoms in ethylene glycol (1,2-glycol), into aldehyde groups, and Hill reagents. (Schiff's Reagent), a fuchsin sulfite dye, reacts in magenta or purplish red, indicating neutral mucopolysaccharide protein.
product content
Cleaning solution (Reagent A) 100 ml
Acid solution (Reagent B) 10 ml
Staining solution A (Reagent C) 10 ml
Oxidizing solution (Reagent D) 10 ml
Staining Solution B (Reagent E) 10 ml
Product manual 1 copy
storage method
Store at room temperature; Reagent C and Reagent E, strictly seal the bottle mouth to avoid light; the reagent is corrosive, pay attention to operation safety; effectively guarantee June
User-supplied
Universal hematoxylin counterstaining kit (80067.1): used for counterstaining after routine staining
Optical microscope: used for observation and analysis after cell staining
Experimental procedure
Operation one: cell slide staining
First, the sample Al New Blue dyeing treatment
- Take out the cell slide or cell smear to be tested
- Carefully add 200 μl of cleaning solution ( Reagent A ) onto the slide or cell smear to cover the entire sample surface.
- Carefully remove the cleaning solution from the slide or cell smear ( Reagent A )
- Carefully add 200 μl of Acid Solution ( Reagent B ) to cover the entire surface of the slide or cell smear sample
- Incubate for 3 minutes at room temperature
- Carefully remove the acid solution from the slide or cell smear ( Reagent B )
- Carefully add 200 μl of cleaning solution ( Reagent A ) onto the slide or cell smear to cover the entire sample surface.
- Carefully remove the cleaning solution from the slide or cell smear ( Reagent A )
- Repeat experiment steps 7 through 8
- Carefully add 200 μl of staining solution A ( Reagent C ) to cover the entire surface of the slide or cell smear sample
- Incubate for 30 minutes at room temperature; or until visible blue, avoid light
- Carefully remove the staining solution A ( Reagent C ) from the slide or cell smear
- Carefully add 200 μl of cleaning solution ( Reagent A ) onto the slide or cell smear to cover the entire sample surface.
- Carefully remove the cleaning solution from the slide or cell smear ( Reagent A )
- Repeat experiment steps 13 through 14
Second, the sample periodic acid treatment
- Carefully add 200 μl of oxidizing solution ( Reagent D ) to cover the entire sample surface
- Incubate for 10 minutes at room temperature
- Carefully remove the oxidizing solution ( Reagent D ) from the slide or cell smear
- Carefully add 200 μl of cleaning solution ( Reagent A ) onto the slide or cell smear to cover the entire sample surface.
- Carefully remove the cleaning solution from the slide or cell smear ( Reagent A )
- Repeat experiment steps 4 to 5
- Carefully add 200 μl of staining solution B ( Reagent E ) to cover the entire surface of the slide or cell smear sample
- Incubate for 10 minutes at room temperature; or until dark red is visible, avoiding light
- Carefully remove the staining solution B ( Reagent E ) from the slide or cell smear
- Carefully add 200 μl of cleaning solution ( Reagent A ) onto the slide or cell smear to cover the entire sample surface.
- Carefully remove the cleaning solution from the slide or cell smear ( Reagent A )
- Repeat experiment steps 10 through 11
Third, (selection step) sample counterstaining treatment
- Sample counterstaining treatment (recommended use of general hematoxylin counterstaining kit-80067.1)
- Put a cover slip or cover
- Immediately observed under a general optical microscope:
Acid mucopolysaccharide protein - bright blue
Neutral mucopolysaccharide protein and other glycoproteins - magenta
Mixed mucopolysaccharide protein - blue or purple
Nucleus - dark blue (multi-dyed)
Operation 2: 24 well cell culture plate staining
First, the sample Al New Blue dyeing treatment
- Carefully remove the medium from the 24-well cell culture plate
- Carefully add 400 μl of cleaning solution ( Reagent A ) to clean the growing cell surface
- Carefully remove the cleaning solution ( Reagent A )
- Carefully add 400 μl of Acid Solution ( Reagent B ) to cover the entire growth surface.
- Incubate for 3 minutes at room temperature
- Carefully remove the acid solution ( Reagent B )
- Carefully add 400 μl of cleaning solution ( Reagent A ) to clean the cell surface
- Carefully remove the cleaning solution ( Reagent A )
- Repeat experiment steps 7 through 8
- Carefully add 400 μl of staining solution A ( Reagent C ) to cover the entire sample surface.
- Incubate for 30 minutes at room temperature; or until visible blue, avoid light
- Carefully remove the stain A ( Reagent C )
- Carefully add 400 μl of cleaning solution ( Reagent A ) to cover the entire sample surface.
- Carefully remove the cleaning solution ( Reagent A )
- Repeat experiment steps 13 through 14
Second, the sample periodic acid treatment
- Carefully add 400 μl of oxidizing solution ( Reagent D ) to cover the entire surface of the sample.
- Incubate for 10 minutes at room temperature
- Carefully remove the oxidizing solution ( Reagent D )
- Carefully add 400 μl of cleaning solution ( Reagent A ) to cover the entire sample surface.
- Carefully remove the cleaning solution ( Reagent A )
- Repeat experiment steps 4 to 5
- Carefully add 400 μl of staining solution B ( Reagent E ) to cover the entire surface of the sample.
- Incubate for 10 minutes at room temperature; or until dark red is visible, avoiding light
- Carefully remove the staining solution B ( Reagent E )
- Carefully add 400 μl of cleaning solution ( Reagent A ) to cover the entire sample surface.
- Carefully remove the cleaning solution ( Reagent A )
- Repeat experiment steps 10 through 11
Third, (selection step) sample counterstaining treatment
- Sample counterstaining treatment (recommended use of general hematoxylin counterstaining kit-80067.1)
- Immediately observed under a general optical microscope:
Acid mucopolysaccharide protein - bright blue
Neutral mucopolysaccharide protein and other glycoproteins - magenta
Mixed mucopolysaccharide protein - blue or purple
Nucleus - dark blue (multi-dyed)
Precautions
- This product is operated 50 times (cell slide) and 25 times (1 24 well plate)
- Wear gloves when handling
- All operations are performed at room temperature
- Avoid using formaldehyde and acetaldehyde alone; use Bouin Fixative - 12267
- Reagents can be reused
- Reagents are corrosive, pay attention to operational safety
- Reagent solution to avoid bacterial contamination
- Precipitation of staining solution B (Reagent E) , can be used after warming, stirring or filtering
- The dyeing liquid B (Reagent E) must be sealed from light, and if it turns red, it will affect the dyeing effect.
- When the reagent solution is on the surface of the sample, avoid the presence of air bubbles and ensure that the sample surface is covered.
- The sample is dyed to avoid excessive, and the dark blue or deep red color can be seen by the naked eye to terminate the dyeing.
- If cytosolic glycoprotein is detected, sample pretreatment is required, such as: formaldehyde vaporization; or anhydrous methanol for 15 minutes; or microwave heating for 45 seconds.
- Enhance stain contrast by using the universal hematoxylin counterstaining kit (80067.1)
- Immediately after the cell staining, optical microscopy
- The company provides a series of glycoprotein analysis reagent products
Quality Standard
- This product has been certified to be stable.
- This product has been identified and clearly colored
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