La detección de glucógeno en las células mononucleares de sangre periférica con ácido periódico de Schiff tinción

The overall goal of this procedure is to visualize the glycogen content of peripheral blood mononuclear cells. This is accomplished by first isolating P BMCs from human whole blood using density gradient centrifugation. The second step is to prepare sample slides by smearing or pipetting the blood cells, and then treating them with fixative solution, followed by washing next for a negative control.

Half of the slide is immersed in amylase solution, which dissolves glycogen polymers, followed by a wash. Now the samples are stained with periodic acid solution followed by a wash. The final step is to add the shift’s reagent followed by a wash.

Ultimately, light microscopy is used to show the glycogen stores inside the cells. The main advantage of this technique over existing methods is that there are more mononuclear cells per slide as compared to whole blood staining. Thus, it’s more efficient to analyze glycogen content and lymphocytes since neutrophils and red blood cells are absent.

To begin this procedure in a biosafety cabinet, carefully transfer 10 to 15 milliliters of whole blood from the heparinized blood collection tube into a 50 milliliter sterile conical tube. Next, dilute the blood in PBS one X at pH 7.4 at a ratio of one to one, and ensure that the maximum total volume does not exceed 30 milliliters. Mix it gently using a serological pipette gun and avoid generating bubbles.

Then transfer 13 milliliters of non ionic synthetic polymer of sucrose, FICO to a new 15 milliliter conical tube. Subsequently, add the diluted blood with slow and steady pressure in order to form a blood layer on top of the FICO layer. Perform this step several times until all the blood has been transferred.

Subsequently, cap the tube tightly and centrifuge it at room temperature for 30 minutes at 400 Gs.After that, slowly take the tube out and transfer it back to the biosafety cabinet. Next, carefully collect the buffy coat between the PBS plasma and FICO layers where the pbmc are located. Avoid collecting the FICO layer and do not disturb the red blood cell layer.

Then transfer the buffy coat into a new 50 milliliter sterile conical tube. Add PBS to the tube with the P BMCs and fill it up to the 45 milliliter mark. Subsequently shake the tube thoroughly.

Then centrifuge it for 15 minutes. At 277 gs, a pallet of p BMCs will be formed at the bottom of the conical tube. Afterward, discard the supernatant into a plastic beaker with 10 milliliters of bleach.

Loosen the cell pellet by gently racking the tube against an undulated surface. Add 20 milliliters of fresh PBS to the tube. If more than one tube is being processed, pull the pellets together in this step.

Next, centrifuge the tube for 12 minutes at 480 gs. Discard the supernatant into the plastic beaker with a splash of bleach. Then gently rack the tube against an undulated surface.

Again afterward, add 25 milliliters of fresh PBS to the tube. Transfer 50 microliters of the cells into a micro centrifuge tube. For viability, count to count the cells.

Add 50 microliters of trian blue and pipette up and down gently to mix. Subsequently, transfer 10 microliters of the mixture to a hemo cytometer. To check the viability of cells and record the number of cells per milliliter.

Take the desired amount of cells and centrifuge the tube for 12 minutes at 177 gs. After that, discard the SUP natant into the beaker with bleach. Then gently rack the tube against an undulated surface, followed by adding 80 microliters of PBS into the tube.

To make the PBMC slide place 80 microliters of the cells onto a pre-labeled microscope slide smear the drop with the help of another slide or place two drops of 40 microliters on both ends of the slide. Leave the slide in the biological safety cabinet to dry. Then prepare the fixative solution by mixing 0.5 milliliters of 37%formaldehyde with 4.5 milliliters of 99%ethanol.

After the slides have dried, add two milliliters of the freshly made fixative solution on the slide to cover the entire surface. Leave the solution on the slide for one minute before rinsing it with tap water for another minute. Then leave it to air dry.

Now dissolve. Point 25 grams of amylase powder in 50 milliliters of distilled water. Pour the solution into a 100 milliliter beaker.

Next, immerse half of the slide in the beaker and incubated for 15 minutes. At room temperature, make a note on the side that is receiving the amylase treatment. Draw a line on the back of the slide indicating the border between the treatment and control.

After that, wash the slide with distilled water to remove the amylase solution. Then leave the slide to air dry In this step, place a slide on a flat surface. Apply two milliliters of periodic acid solution on the sample and incubate it for five minutes at room temperature.

Then rinse the slide several times with distilled water. Apply two milliliters of shifts reagent on the slide and incubate it at room temperature for 15 minutes. Subsequently, wash the slide with distilled water for five minutes and then leave it to air dry.

Next, apply 100 microliters mounting media on the slide and cover it with one large cover slip, or apply 50 microliters and use two small cover slips. After that, apply clear nail polish on the edges of the cover slip. Let them dry overnight.

Then obtain images with the binocular light microscope using the 100 x objective PAS. Staining was performed on P BMCs from the venous blood of healthy subjects. The PAS stained P BMCs displayed a variety of staining patterns.

Small cells with granules were readily observed. A proportion of larger cells with a diffused staining pattern were also observed. Treatment of samples with amylase removed the granule signal and diminished the diffused PAS positivity.

The PAS and HEMATIN staining were performed on the whole blood slide. This PBMC is surrounded by many erythrocytes. 98%PAS positive pbmc were the smaller cells, and 40%were the larger cells.

Amylase treatment eliminated the PAS signal in the small cells and significantly diminished the PAS signal in the larger cells to 7%Don’t forget that working with PAS reagents can be extremely hazardous, and precautions such as wearing appropriate personal protection equipment and using a chemical hood should be taken while performing this procedure.