The overall goal of this procedure is to analyze the expression of proliferation markers of interest by immunocytochemical and histological analyses of thromboplastin-plasma cell block embedded tissue clumps. This method can help answer the key question about the clinical pathology of cell blocking by the tissues. The main advantage of this alternative method for immunocytochemical analysis of paraffin embedded cell blocks is the technical simplicity of the procedures.

When a 100 millimeter dish of HeLa cell culture reaches confluency, replace the supernatant with 10 milliliters of HeLa cell culture medium without FBS and return the dish to the cell culture incubator. After 48 hours, wash the cells with two milliliters of PBS followed by incubation in two milliliters of 0.25%EDTA plus trypsin for two to three minutes at 37 degrees celsius and 5%carbon dioxide. When the cells have detached, stop the reaction with five milliliters of complete medium and transfer the cell solution into a 15 milliliter conical tube.

Collect the cells by centrifugation followed by two washes in two milliliters of cold PBS per wash. After the second wash, replace the supernatant with one milliliter of 95%ethanol and mix the pellet by vortexing. Then place the fixed cells on ice.

To prepare a paraffin cell block, after collecting EDTA plasma from healthy donor blood, centrifuge the samples and transfer 200 to 400 microliter supernatant plasma aliquots into individual microfuge tubes. Next add about 200 microliters of plasma, about 200 microliters of thromboplastin, and about 200 microliters of 025 molar calcium chloride to the fixed HeLa cells. Allow the mixtures to form cell clots at room temperature for ten minutes, then wash the clots two times with one milliliter of PBS fully decanting the clots onto individual pieces of formalin moistened filter paper after the second wash.

Wrap the clots in the filter paper and use a pin set to place the cloths into individual tissue cassettes in the center of four other pieces of formalin moistened paper. Then place the tissue cassettes into glass jars containing 50 milliliters of buffered formalin for overnight formalin fixation at four degrees celsius. The next morning load the cassettes into a tissue processor for overnight water removal and fixed cell conditioning.

At least one hour before the end of the processing procedure, turn on a heated embedding station to melt the paraffin. When the embedding station and the clot are ready, confirm the presence of molten paraffin in the metal mold and transfer a formed cell clot into the paraffin. Place a new tissue cassette without the lid into the metal mold and cover the cassette with more molten paraffin.

Let the paraffin solidify in a cold plate for 30 to 60 seconds. Then separate the tissue cassette from the metal mold. To prepare sections for immunocytochemical analysis, locate the cell clot in one paraffin cell block and use a microtome to cut the block into three to four micrometer thick slices.

Place paraffin sections onto saline coated glass slides and place the slides into a 37 degree celsius oven for 30 minutes. When the sections have adhered to the slides, de-paraffinize the slides in 15 milliliters of xylene for four minutes followed by dehydration of the sections with sequential two minute descending ethanol incubations. After the 80%ethanol incubation, rinse the sections in running water for 10 minutes and boil the slides in a jar containing 40 milliliters of tris-EDTA retrieval buffer for 30 minutes.

At the end of the incubation, wash the antigen retrieved slides under running water followed by a 10 minute incubation in 95%ethanol at four degrees celsius. After air drying, use a hydrophobic pen to encircle the cell staining area on each slide. Wash the slides in TBS-T followed by incubation in hydrogen peroxide block for 15 minutes at room temperature to remove any remnant peroxidase activity.

Wash the slides three times in TBS-T for two minutes each wash, then label the sections with 100 microliters of primary antibody mixture from the immunocytochemical stain kit of interest for one hour followed by five two minute TBS-T washes. After the last wash, incubate the slides in primary antibody enhancer from the kit for 15 minutes at room temperature in the dark. At the end of the incubation, wash the sections four times in TBS-T adding about 200 microliters of secondary antibody labeled with horseradish peroxidase after the last wash for a 30 minute incubation at room temperature.

Wash the enhanced sections five times in fresh TBS-T followed by the addition of 100 microliters of diaminobenzidine solution per section for three minutes. Wash the slides two times in TBS-T and label the sections with 100 microliters of hematoxylin solution for one minute. Then wash the slides one more time in TBS-T and incubate the slides in 95%ethanol for two minutes followed by one dip in fresh 95%ethanol and two dips in 100%ethanol.

Incubate the ethanol dehydrated sections in 40 milliliters of xylene in a glass jar for five minutes and allow the slides to air dry. Then mount a cover slip onto each slide and observe the samples under a light microscope. Hematoxylin and eosin staining of the paraffin embedded tissue as just demonstrated reveals mostly in tact nuclei and cytoplasm suggesting an excellent morphological preservation of the cell samples by this method.

Poorly prepared cell blocks, however, exhibit a poor morphology and irregular labeling even if the samples are stained properly. Cytoskeleton associated protein two is typically observed within the condensed chromatin, mitotic spindle, and cytoplasm. Only the cells with cytoskeleton associated protein two staining in the condensed chromatin are mitotic cells whereas few cytoskeleton associated protein two positive cells are found within the serum starved HeLa cell culture population.

Most of the highly mitotic HeLa cells are also Ki-67 positive and Ki-67 staining is seen in the cell nuclei. Only about half of the serum starved HeLa cells express this proliferation marker. Once mastered, this technique can be completed in six hour if it is performed properly.

While attempting this procedure it's important to remember to dilute the cells to an appropriate density for making a good clot. Following this procedure other method like flow cytometric analysis of the fixed cells can be performed to answer additional question about the cell cycle phase during synchronization. After its development, this technique paved the way for a future in the field of the liver pathology to explore expression profiling in cell line while preserving morphological information in cultured cells.

After watching this video you should have a good understanding of how to perform immunocytochemistry and to prepare plasma thromboplastin blocks from the cultured cells. Don't forget that working with hazardous reagents like xylene can be extremely hazardous and that precautions such as wearing gloves and a lab coat should always be taken while performing this procedure.

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