Experiments on the display of sugars in cells
Experiments on the display of sugars in cells
Polysaccharides, mucopolysaccharides and mucins in animal tissues are generally shown by PAS.
Principle
The basic principle of the PAS method for the visualization of sugars in cells is that periodic acid is an oxidizing agent that breaks C-C bonds within various structures. Sugars containing glycol groups (CHOH-CHOH) are oxidized in the presence of periodic acid to produce a double aldehyde group (CHO-CHO), and the free aldehyde group reacts with colorless magenta in Schiff's reagent to produce a purplish-red compound that adheres to the sugar-containing tissue. Periodic acid is superior to other reagents commonly used in histology to oxidize the C-C bond ( KMnO4, H2CrO4, H2O2 ) because it does not further oxidize the resulting aldehyde group, which can be localized by reacting with Schiff's reagent to produce a purplish-red compound.
Operation method
PAS method to show sugars in cells
Principle
The basic principle of the PAS method for the visualization of sugars in cells is that periodic acid is an oxidizing agent that breaks C-C bonds within various structures. Sugars containing glycol groups (CHOH-CHOH) are oxidized in the presence of periodic acid to produce a double aldehyde group (CHO-CHO), and the free aldehyde group reacts with colorless magenta in Schiff's reagent to produce a purplish-red compound that adheres to the sugar-containing tissue. Periodic acid is superior to other reagents commonly used in histology to oxidize the C-C bond (KMnO4, H2CrO4, H2O2) because it does not further oxidize the resulting aldehyde group, which can be localized by reacting with Schiff's reagent to produce a purplish-red compound.
Materials and Instruments
Equipment: Move The basic process of PAS method to show the sugars in cells can be divided into the following steps: (I) Tissue section Caveat 1. The degree of staining depends on the duration of the period of treatment with periodate. Therefore, the section should not be treated in aqueous periodic acid for too long. 2. After PAS reaction, due to the oxidation of periodic acid to accelerate the staining of hematoxylin, its staining time can be appropriately reduced, commonly used in hematoxylin diluted staining solution re-staining. For more product details, please visit Aladdin Scientific website.
Paraffin slicer, microscope, routine laboratory instruments, liver, kidney, cardiac muscle, skeletal muscle or other tissues from animals.
Reagents:
① 1% periodic acid
② Schiff's reagent
（Schiff's reagent (alkaline magenta, 1 g; distilled water, 200 mL; 1 mol/L HCI, 20 mL; sodium metabisulfite, 1.0 g; activated carbon, 2.0 g.). Dissolve 1 g of basic magenta in 200 mL of boiling distilled water, shake for 5 min to dissolve, and cool to 50 °C. Filter and add 20 mL of 1 mol/L HCl to the filtrate, cool to 25 ℃, add 1.0 g of sodium metabisulfite, and let it stand in the dark at room temperature for 24 h. Add 2.0 g of activated carbon, 2.0 g of activated carbon, and 2.0 g of activated carbon. Add 2.0 g of activated carbon and shake for 1 min. Filtration. Place in a brown bottle sealed, stored at 4 ℃.)
③ 0.5% sodium metabisulfite solution
(10% sodium metabisulfite, 5 mL; 1 mol ／ L HCl, 5 mL; distilled water, 90 mL. Mix well before use.)
④Acetic anhydride-pyridine mixture
(Acetic anhydride, 16 mL; pyridine (anhydrous), 24 mL)
⑤ 1% Starch Glycosylase Solution
⑥ Carnoy's fixing solution
(7) Hematoxylin staining solution (Hematoxylin, 2.0 g; 95% ethanol, 100 mL; glacial acetic acid, 10 mL; pure glycerol, 100 mL; potassium alum, 3.0 g; distilled water, 200 mL). The specific preparation method is as follows: ① dissolve hematoxylin in 25 mL of 95% ethanol and glacial acetic acid, and then add glycerol and the remaining ethanol; ② dissolve potassium alum in water, and heat to dissolve; ③ add the potassium alum solution into the hematoxylin solution slowly, stirring as it is added, and then leave it for about 3 weeks at a bright place after mixing; ④ filter, and standby).
A. Take 1~2 mm thick liver, kidney, cardiac muscle, skeletal muscle or other tissue blocks, fixed with Carnoy's fixative, and put it in the refrigerator at 4 ℃ for 2~4 h.
B. The specimen was dehydrated by 90%, 95%, and 100% ethanol for three times, and then transparent in xylene, and embedded in paraffin wax.
C. The sections were deparaffinized in distilled water.
D. Wash in 1% aqueous peroxodisulfuric acid for 2~5 min. E. Wash in distilled water. D. Into 1% periodic acid aqueous solution for 2-5 min.
E. Wash with distilled water.
F. Soak with Schiff's reagent for 15 min.
G. Soak with 0.5% sodium metabisulphite solution 3 times for 2 min each time.
H. Rinse with running water for 5 min, and soak with distilled water for 1 min.
I. Re-dyeing the nuclei with hematoxylin.
J. Rinse with running water for 5 min, and then pass through distilled water, and then absorb.
K. Dehydrate with 95% and 100% ethanol 2 times. L. Dehydrate with 100% ethanol twice.
L. Xylene clear, neutral gum sealing.
2. Photo 1 (blocking the PAS reaction by acetylation)
A. Treat the photos with acetic anhydride-pyridine mixture at 22 ℃ for 1-24 h.
B. Wash with water.
C. Carry out the PAS reaction mentioned above.
3. Photo 2 (starch glycolysis enzyme-treated sections)
A. Deparaffinize the sections to water, and treat the slices with 1% amylose glycolysis enzyme solution (pH 6.0) for 40 min (at room temperature) at 37 ℃. A. Treat the sections with 1% amylase solution (pH 6.0) at 37℃ for 40 min (60 min at room temperature); or treat with saliva for 60 min at room temperature (30 min for two times).
B. Wash the sections in running water for 5~10 min, and then wash with distilled water.
C. Perform the PAS reaction as described above.