Periodic acid-Schiff's reaction experiment

Summary

The principle of PAS reaction to show sugars is due to the polysaccharides containing glycol groups, under the action of periodic acid, oxidation to produce aldehyde groups, this aldehyde plug and then with the action of the Schiffs solution will be colorless magenta into violet-red dye and attached to the tissues containing polysaccharides. This experiment is from Mudanjiang Medical College, undergraduate 5-year laboratory guide for testing majors.

Operation method

Periodic acid-Schiff's reaction experiment

Principle

The principle of the PAS reaction to show sugars is due to the polysaccharides containing glycol groups, under the action of periodic acid, oxidation produces aldehyde groups, this aldehyde plug and then with the action of the Schiffs solution will be colorless magenta into a purplish-red dye and attached to the tissues containing polysaccharides. Basic magenta + 2H2SO3 → magenta sulfite (colorless) magenta sulfite + with aldehyde group → colorless material

Materials and Instruments

Specimens
Basic compound red HCI Activated charcoal Acid alcohol
Smear

Move

I. Experimental reagents:

1. Specimen selection: generally can be used for fresh and dry smears, unstained and long-term storage of the specimen can also be used. If the specimen is stained with Rachel's method, it can be decolorized with acid and alcohol first and then stained with PAS.

Schiff's solution.

2. Preparation: alkaline compound red 1 g dissolved in 200 ml of boiling water, cooled to 60 ℃ for filtration, after filtration, add 20 ml of 1N HCI and then add NaHSO32 grams (more can also be added), in order to have a strong pungent taste of SO2 is good, tightly closed bottle filtration, the liquid was a light tea color can be used. Then add 300mg of activated carbon filtration, the filtrate should be transparent and colorless. Stored in the refrigerator at 4 ℃.

Second, the experimental steps

1. Dry blood or bone marrow slices are immobilized in 95% alcohol for 10 minutes.

2. Rinse several times with distilled water

3. 1% periodic acid (aqueous solution) for 10 minutes (for 1-2 weeks)

4. Several rinses with distilled water

5. Place in Schiff's solution for 30 minutes

6. rinse with tap water (about 5 minutes) and finally rinse with distilled water, then restain with 2% methyl green for 20 minutes.

Integral labeling: (blood film counts small lymphocytes)

"0" No intracytoplasmic granules

+ A ring of PAS-positive granules in the cytoplasm

++ Two rings of PAS-positive particles in cytoplasm.

+ + + + Three rings of PAS-positive particles in cytoplasm, or large clumps formed

Count 100 cells to find the integral

Normal value: (blood film lymphocytes)

Positive rate 10-30%, score 12-56, mean 31

Nucleated red blood cell integral method:

"0" positive

"+" A small number of positive particles dispersed in the cytoplasm or a light red color, which should be darker than normal erythrocytes.

"++" One or two dense granular rings in the cytoplasm or a moderately diffuse red color of the cytoplasm

"+ + + +" There are a large number of dense particles in the cytoplasm, or a very dark red color.

Normal values: Nucleated Red Score 0-7 mostly negative.

Caveat

1. Schiff's solution turns red and can no longer be used.

2. The degree of saturation of SO2 in Schiff's solution is related to the constancy of PAS reaction, NaHSO3 will be ineffective for a long time, and SO2 will not be saturated, so NaHSO3 can be added more.

3， alkaline magenta factory grade and dyeing depth, add NaHSO3 overnight is still red, then the dye is not suitable. 4, activated carbon can be added more, and PAS reaction constancy is related.

4. activated carbon can be added more, the purpose is to adsorb the color, such as the Department of magenta is not suitable for more activated carbon color can not be adsorbed.

5. The apparatus for preparing Schiff's liquid should be very clean and dry.

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