This work was supported by research grants to K.-M.H. from the National Cancer Center, Korea (1510121) and National Research Foundation, Korea (no. NRF-2015R1A2A2A04007432).

The study protocol was approved by the Institutional Review Board of National Cancer Center, Korea (NCCNCS-12-630).
1. Sample Preparation (30 min)
<ol>
	<li>For the culture of HeLa cells (CCL-2, ATCC), use 10 mL complete DMEM medium (10% fetal bovine serum, 1% pen-strep) in a 100 mm culture dish. To prepare serum-starved HeLa cells, incubate confluent HeLa cells in DMEM without fetal bovine serum for 48 hours, as described in a previous study11.</li>
	<li>To detach the cells, wash them with 2 mL phosphate-buffered saline (PBS), and add 2 mL 0.25% EDTA trypsin per 100 mm culture dish. Then, incubate for 2 - 3 min in a CO2 incubator at 37 &#176;C.</li>
	<li>Add 5 mL of complete DMEM medium to the culture dish to deactivate trypsin, and transfer the detached cells to a 15 mL tube. Then, centrifuge the cells at 300 x g for 10 min to form a pellet.</li>
	<li>Remove the supernatant, and wash the pellet twice with 2 mL cold PBS by centrifugation at 300 x g for 10 min.</li>
	<li>After removal of the supernatant, add 1 mL of 95% ethanol to the cell pellet, and mix the cell pellet with ethanol by vortexing. Keep the fixed cells on ice until the cell-block preparation.</li>
</ol>
2. Cell-block Preparation (1 h 30 min)
<ol>
	<li>For the preparation of frozen plasma aliquots, collect EDTA plasma from healthy donor blood and centrifuge at 13,000 x g for 10 min. Collect the supernatant plasma, and aliquot 200 - 400 &#181;L each into microfuge tubes. Store the aliquots at -80 &#176;C until use. When the frozen plasma aliquots are ready, retrieve them and thaw the plasma by incubation at 37 &#176;C for 5 min.</li>
	<li>Centrifuge the fixed cells at 700 x g for 10 min, then discard the supernatant.</li>
	<li>Mix the cell pellet with 2 drops (about 200 &#181;L) of the plasma, 2 drops of thromboplastin, and 2 drops of 0.025 M calcium chloride. Then, mix them by pipetting.</li>
	<li>Allow the mixture to form a cell clot at room temperature for 10 min, as shown in Figure 2A. Then, wash the cell clot with PBS two times by pouring and removing PBS by pipette. The representative white cell clot is shown in Figure 2B.</li>
	<li>Moisten a piece of filter paper with formalin, and wrap the cell clot with the pre-moistened filter paper. Then, place the clotted mixture with a pincette in a tissue cassette as shown in Figure 2C.</li>
	<li>Place the tissue cassette in a glass jar containing 50 mL of buffered formalin (10% formalin in PBS) overnight at 4 &#176;C for formalin fixation.</li>
</ol>
3. Tissue Processing and Paraffin Embedding (Overnight)
<ol>
	<li>Load the tissue cassette containing the formalin-fixed cell clot in a tissue processor overnight as described previously11. The tissue processing is for water removal and conditioning of fixed cells prior to paraffin embedding. For research without tissue processing, perform the tissue processing procedure as shown in Table 1.</li>
	<li>Turn on the heated embedding station, and 60 minutes later, check that there is molten paraffin in the metal mold therein (Figure 2D).</li>
	<li>Take the formed cell clot from the tissue cassette and place it in the molten paraffin within the metal mold.</li>
	<li>Place the new tissue cassette without the lid into the metal mold containing the cell clot in the middle of the molten paraffin, and pour more molten paraffin into the tissue cassette and onto the metal mold. Then, let the paraffin solidify in the cold plate for 30 - 60 seconds.</li>
	<li>Separate the tissue cassette from the metal mold. Then, the paraffin cell block is ready for immunocytochemistry. The embedded cell clot in the paraffin-embedded cell block is indicated by the arrow in Figure 2E.</li>
</ol>
4. Preparation of Slides for Immunocytochemistry (1 h)
<ol>
	<li>Check the area for the cell clot in the paraffin cell block, and cut the cell block into slices with thicknesses of 3 - 4 &#181;m using a microtome. Put the paraffin sections on silane-coated glass slides, as shown in Figure 2F.</li>
	<li>Place the section slides in a 37 &#176;C oven for 30 min to make the sections adhere to the slides.</li>
</ol>
5. Immunocytochemistry of Cell Blocks (6 h)
NOTE: For immunocytochemical staining on cell-block sections, various kits can be used (see Table of Materials). All such kits have various sensitivities and specificities depending on their modifications.
<ol>
	<li>Incubate the sections in 50 mL of xylene for 4 min to de-paraffinize.</li>
	<li>Incubate the slides in 100% ethanol for 2 min for dehydration, and two times in 95% ethanol, and in 80% ethanol for 2 min. Then, remove the ethanol in running water for 10 min.</li>
	<li>For antigen retrieval, place the slides in a jar containing 40 mL Tris-EDTA retrieval buffer, pH 9.0, and boil in a cooker for 30 min.</li>
	<li>Wash the slides under running water, and incubate them in 95% ethanol for 10 min at 4 &#176;C. Then, mark the cell-staining area on the slides with a Pap pen for easy identification.</li>
	<li>Wash the slides in Tris-buffered saline containing 0.2% Tween 20 (TBS-T), and incubate in the hydrogen peroxide block (see Table of Materials) at room temperature for 15 min to remove any remnant peroxidase activity. Then, wash the slides in TBS-T three times for 2 min each.</li>
	<li>Prepare diluted antibody solution by diluting primary antibody in the protein block (see Table of Materials). For example, the CKAP2 primary antibody can be diluted 1:100, and the Ki-67 antibody 1:500. Then, incubate the slides in 100 &#181;L of diluted antibody solution for 1 h.</li>
	<li>After washing in TBS-T five times for 2 min each, incubate the slides in the primary antibody enhancer in the kit for 15 min at room temperature in the dark.</li>
	<li>After the washing in TBS-T four times for 2 min each, add 2 drops of HRP polymer (a secondary antibody labeled with horseradish peroxidase), and incubate the slides at room temperature for 30 min.</li>
	<li>After washing in TBS-T five times for 2 min each, add 100 &#181;L of diaminobenzidine (DAB) solution (see Table of Materials), and incubate the slides for 3 min.</li>
	<li>After washing in TBS-T two times, add 100 &#181;L of Hematoxylin solution (the mixture of 100 &#181;L of Hematoxylin and 600 &#181;L of distilled water), and incubate the slides for 1 min.</li>
	<li>After washing the slides in TBS-T once, dehydrate by incubating in 95% ethanol for 2 min, dipping in 95% ethanol once, and dipping in 100% ethanol two times. Then, incubate the slides in 40 mL xylene in a glass jar for 5 min.</li>
	<li>When the slides are dry from xylene, mount the respective coverslips.</li>
	<li>Observe the staining patterns using light microscopy.</li>
</ol>

<ol>
	<li>Bhattacharyya, D., Hammond, A. T., Glick, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-quality+immunofluorescence+of+cultured+cells.">High-quality immunofluorescence of cultured cells.</a> Methods Mol Biol. 619, 403-410 (2010).</li><li>Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy+-+Avoiding+the+pit+falls.">Fluorescence microscopy - Avoiding the pit falls.</a> J Cell Sci. 120, 1703-1705 (2007).</li><li>Odell, I. D., Cook, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+Techniques.">Immunofluorescence Techniques.</a> J Invest Dermatol. 133, e4 (2013).</li><li>Kulkarni, M. B., Desai, S. B., Ajit, D., Chinoy, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+the+thromboplastin-plasma+cell-block+technique+for+fine-needle+aspiration+and+serous+effusions.">The utility of the thromboplastin-plasma cell-block technique for fine-needle aspiration and serous effusions.</a> Diagn Cytopathol. 37 (2), 86-90 (2009).</li><li>Shivakumarswamy, U., Arakeri, S. U., Karigowdar, M. H., Yelikar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+utility+of+the+cell+block+method+versus+the+conventional+smear+study+in+pleural+fluid+cytology.">Diagnostic utility of the cell block method versus the conventional smear study in pleural fluid cytology.</a> J Cytol. 29 (1), 11-15 (2012).</li><li>Nathan, N. A., Narayan, E., Smith, M. M., Horn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+block+cytology:+Improved+preparation+and+its+efficacy+in+diagnostic+cytology.">Cell block cytology: Improved preparation and its efficacy in diagnostic cytology.</a> Am J Clin Pathol. 114 (4), 599-606 (2000).</li><li>Keyhani-Rofagha, S., Vesey-Shecket, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+value,+feasibility,+and+validity+of+preparing+cell+blocks+from+fluid-based+gynecologic+cytology+specimens.">Diagnostic value, feasibility, and validity of preparing cell blocks from fluid-based gynecologic cytology specimens.</a> Cancer. 96, 204-209 (2002).</li><li>Young, N. A., Naryshkin, S., Katz, S. 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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CKAP2+(cytoskeleton-associated+protein2)+is+a+new+prognostic+marker+in+HER2-negative+luminal+type+breast+cancer.">CKAP2 (cytoskeleton-associated protein2) is a new prognostic marker in HER2-negative luminal type breast cancer.</a> PLoS ONE. 12 (8), e0182107 (2017).</li><li>Inwald, E. C., Klinkhammer-Schalke, M., Hofstadter, F., Zeman, F., Koller, M., Gerstenhauer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ki-67+is+a+prognostic+parameter+in+breast+cancer+patients:+results+of+a+large+population-based+cohort+of+a+cancer+registry.">Ki-67 is a prognostic parameter in breast cancer patients: results of a large population-based cohort of a cancer registry.</a> Breast Cancer Res Treat. 139 (2), 539-552 (2013).</li><li>Katikireddy, K. 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All authors have declared no conflicts of interest.

Immunofluorescent staining on fixed cultured cells is currently the method of choice for determination of protein expression level in cells while preserving morphological information1. However, immunocytochemistry on paraffin-embedded cell blocks can be an excellent alternative. The detailed procedures for the preparation of paraffin-embedded cell blocks and immunocytochemistry have been described in this protocol, and we hope it can facilitate the application of immunocytochemistry in cell studies.
Immunocytochemistry has several advantages over immunofluorescent staining. Immunofluorescent staining for cells usually requires freshly cultured cells, but paraffin cell blocks can be kept at room temperature for several years13. Additionally, immunocytochemistry on cell blocks can explore intracellular expression patterns by employing the same antibody used in routine immunohistochemistry on human tissues4. Furthermore, it can explore the changes in protein levels or posttranslational modifications either by pre-incubating cells with various drugs or under various culture conditions11.
In contrast to the advantages of immunocytochemical staining, the preparation of paraffin-embedded cell blocks takes time and is costly14. Also, most research laboratories lack experience in this technique, and technical errors under such circumstances are common. The most common errors are poor preservation of cell morphology and poor or irregular immunocytochemical staining on the paraffin cell block sections. These and most others can be avoided by making cell blocks under the best cell conditions and using enough solution to form cell clots.
As a demonstration of the present protocol, cell blocks were prepared for HeLa cells, and immunocytochemical staining was performed for two proliferation markers, CKAP2 and Ki-67, as previously reported11. For immunocytochemistry, the cells were manipulated by incubation in media with and without fetal bovine serum, and the effect of serum starvation could be observed. These prepared paraffin-embedded cell blocks can be employed for a large number of antibodies, because many slides can be prepared from a cell block using only a 4 - 5 &#181;m-thick cell-block section per slide. Therefore, expression patterns corresponding to two different conditions can be evaluated with several different antibodies. The immunostaining patterns for CKAP2 and Ki-67 in cancer tissues have already been reported9,10,11,12, and the immunocytochemical staining results could be easily evaluated, because the staining patterns were quite similar to those from immunohistochemistry.
In conclusion, immunocytochemical staining on paraffin cell blocks can be an excellent alternative to immunofluorescent staining; moreover, it can be easily and reliably employed in basic research for expression profiling in cell lines while preserving morphological information.

In most laboratories, paraffin-embedded cell blocks are not commonly used. Rather, fixed cultured cells, not paraffin-embedded cells, are employed in subcellular localization studies. For those fixed cultured cells, fluorescence instead of chromogen has been used. Therefore, immunofluorescent staining is currently the method of choice for determination of protein expression levels in research employing cell cultures1. Slides prepared for immunofluorescent staining, however, can be observed only under immunofluorescent microscopy, which might show images quite different from those indicated under plane microscopy2. Additionally, preservation of slides for immunofluorescent staining requires protection from bright light, and fluorescent signals become weaker with repeated exposure for imaging due to the loss of the fluorescent signal3. Results from immunocytochemistry on paraffin-embedded cell blocks are quite similar to those from immunohistochemistry on paraffin-embedded tissues4, and they can easily be translated into clinical information. Therefore, immunocytochemistry can be an excellent alternative. However, cell-block preparation has not been popular in basic research laboratories. In this protocol, then, cell-block preparation and immunocytochemical staining are provided to promote the use of this method in the field of cell-culture studies.
Cell-block preparation and immunocytochemistry are not unique methods, and they have already been applied from clinical diagnosis to basic research4,5. Although various cell-block preparation methods have been reported4,6 the thromboplastin-plasma method is simple, cost-effective, and readily adaptable. Therefore, in the protocol presented in this paper, the thromboplastin-plasma method4,5,6,7,8 is used for preparation of paraffin-embedded cell blocks.
In the present study, the detailed procedures for the preparation of thromboplastin-plasma cell blocks and the immunocytochemistry method employing two proliferation markers were demonstrated. One marker is cytoskeleton-associated protein 2 (CKAP2), which has recently been reported as a mitotic marker9,10,11; the other one is Ki-67, which is the best-known proliferation marker12. The representative scheme is shown in Figure 1.

<table border="0" cellpadding="0" cellspacing="0" style="width:844px;" width="844">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">HeLa Cells</td>
			<td style="width:221px;">ATCC</td>
			<td style="width:291px;">HeLa (ATCC&#160;CCL-2)</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ki-67 Antibody</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td>RM-9106-S1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Paraffin</td>
			<td style="width:221px;">Leica</td>
			<td style="width:291px;">39601006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Xylene</td>
			<td style="width:221px;">Fisher SCientific</td>
			<td>1330-20-7,100-41-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ethenol</td>
			<td style="width:221px;">GD Chem</td>
			<td style="width:291px;">DJ16016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Hematoxylin</td>
			<td style="width:221px;">Agilent</td>
			<td style="width:291px;">10118581</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">DAB Quanto Kit</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">TA-125-QHDX, QHCX 170405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ultravision LP detection Kit</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">PBQ141209, LPB141209, LPH141210</td>
			<td>Kit for immunocytochemistry; contains protein block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TintoRetriever Pressure Cooker</td>
			<td style="width:221px;">Bio SB Corporation</td>
			<td>BSB 7008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tris-buffered saline</td>
			<td style="width:221px;">iNtRON Biotechnology</td>
			<td style="width:291px;">IBS-BT008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tween 20</td>
			<td style="width:221px;">USB Corporation</td>
			<td style="width:291px;">115106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Thromboplastin</td>
			<td>Neoplastin Cl Plus</td>
			<td>NC0591432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tris-EDTA retrival Buffer</td>
			<td style="width:221px;">Dignostic Biosystem</td>
			<td style="width:291px;">E625-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Trypsin EDTA</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal&#160;bovine serum (FBS)</td>
			<td>Hyclone Laboratories Inc</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Hyclone Laboratories Inc</td>
			<td style="width:291px;">SH30243.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Antibiotic-Antimycotic, 100X</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">15240062</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:261px;">Ultra vision peroxide block H2O2</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">02Q141212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Mounting Medium</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">363313</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Pap-Pen</td>
			<td>Vector Laboratories</td>
			<td style="width:291px;">H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Filter Paper</td>
			<td style="width:221px;">GE Healthcare Life Sciences</td>
			<td style="width:291px;">1001-0155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Calcium Chloride</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:291px;">21115-100ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Phosphate-buffered saline</td>
			<td style="width:221px;">PAA Laboratories</td>
			<td style="width:291px;">H21-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Formalin</td>
			<td style="width:221px;">Daejung</td>
			<td style="width:291px;">50-00-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">15 ml tube</td>
			<td style="width:221px;">SPL Life Sciences</td>
			<td style="width:291px;">50015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">tissue processing cassette</td>
			<td style="width:221px;">Simport</td>
			<td style="width:291px;">M492-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm culture dishes</td>
			<td style="width:221px;">BD Biosciences</td>
			<td style="width:291px;">08-772E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Glass Slide</td>
			<td style="width:221px;">Muto Pure Chemicals</td>
			<td style="width:291px;">140712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Cover Glass</td>
			<td style="width:221px;">Marienfeld</td>
			<td style="width:291px;">2262817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Glass Zar</td>
			<td style="width:221px;">Hyunil Lab-Mate</td>
			<td style="width:291px;">HIP-1027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Centrifuge</td>
			<td style="width:221px;">Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Class II Laminar Flow Hood</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">1300 series A2</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:261px;">CO2 Incubator</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">Series A2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tissue Processor</td>
			<td style="width:221px;">Leica BioSystem</td>
			<td style="width:291px;">TP1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Microtome</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">HM340E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Microscope</td>
			<td style="width:221px;">Olympus</td>
			<td style="width:291px;">CX-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Paraffin embedding station</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">EC 350-1, EC 350-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tissue Section Bath (Round)</td>
			<td style="width:221px;">CellPath</td>
			<td style="width:291px;">HCP-JAW-0100-00AEU</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:261px;">Fume Hood</td>
			<td style="width:221px;">Hanyang Scientific Equipment Co. Ltd.</td>
			<td style="width:291px;">FH-150</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of protein expression level in cultured cells by immunocytochemistry on paraffin-embedded cell blocks

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

In a hematoxylin-and-eosin-stained slide from the paraffin-embedded cell block (Figure 3A,B), most of the nuclei and cytoplasm of the cells are intact, suggesting that the morphological preservation is excellent with the current protocol (Figure 3A). In the immunocytochemical staining, positive CKAP2 staining was observed in condensed chromatin, mitotic spindle, and cytoplasm (Figure 4), as previously reported10. Ki-67 staining was observed in the cell nuclei, as expected (Figure 4). Only the cells with CKAP2 staining in condensed chromatin (see the arrows in Figure 4A,B) were mitotic cells. Many CKAP2-positive cells were shown in the highly mitotic HeLa cells that had been prepared after an incubation in complete medium&#160;(Figure 4A). In comparison, there were few CKAP2-positive cells in the serum-starved HeLa cells (Figure 4B). Most of the highly mitotic HeLa cells were Ki-67 positive (Figure 4C). Contrastingly, the Ki-67-positive rate in the serum-starved HeLa cells remained as high as ~50% (Figure 4D). These results are quite comparable to those of a previous report11, which suggests that CKAP2 is a more reliable proliferation marker in cancer cells than is Ki-67.
In poorly prepared cell blocks, the nuclei are separated from the cytoplasm, and there is also, resultantly, poor morphologic preservation. Longer-than-overnight incubation of fixed cells in a refrigerator might cause such poor results. Another important problem is that cells are not stained well by immunocytochemistry, notwithstanding the excellent morphology. This problem arises more often when the cell clot is small. Typically, the staining intensity is irregular as shown in Figure 3B; but when the cell clot is larger, there is much less chance of irregular staining. Therefore, in this protocol, we increased the volumes of plasma, thromboplastin, and calcium chloride in order to form a large cell clot.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57369/57369fig1.jpg" /> 
Figure 1: Cell-block preparation scheme. <a href="/files/ftp_upload/57369/57369fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57369/57369fig2.jpg" /> 
Figure 2: Illustration of paraffin cell-block preparation. (A) Thromboplastin-plasma cell clot in tube. (B) TP Cell clot after PBS washing. (C) Cell clot on moistened filter paper. (D) Tissue-embedding station with molten wax. Metal mold (arrow) holds molten wax for solidification. (E) Paraffin-embedded cell clot (arrow) embedded in paraffin or paraffin-embedded cell block. (F) Thin paraffin section on the middle part of a coated slide. <a href="/files/ftp_upload/57369/57369fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57369/57369fig3.jpg" /> 
Figure 3: Cell-block preparation and confirmation of quality by hematoxylin and eosin and immunostaining. (A) Hematoxylin-and-eosin-stained image of HeLa cells in a paraffin-embedded cell-block section. (B) Irregular staining of Ki-67 in immunocytochemistry on a poorly prepared paraffin-embedded cell-block section. Scale bars are (A) 100 and (B) 200 &#956;m. <a href="/files/ftp_upload/57369/57369fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57369/57369fig4.jpg" /> 
Figure 4: Immunocytochemical staining on paraffin-embedded cell block for HeLa cells. (A) CKAP2 staining under highly mitotic conditions. (B) CKAP2 staining under serum-starved conditions. (C) Ki-67 staining under highly mitotic conditions. (D) Ki-67 staining under serum-starved conditions. 100 &#956;m scale bars are shown. The arrowheads indicate CKAP2-positive cells. <a href="/files/ftp_upload/57369/57369fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Procedure</td>
			<td>Steps</td>
			<td>Solution</td>
			<td>Time/Temperature</td>
		</tr>
		<tr>
			<td rowspan="4">Dehydration</td>
			<td>1</td>
			<td>70% alcohol</td>
			<td>15 min/RT</td>
		</tr>
		<tr>
			<td>2</td>
			<td>80% alcohol</td>
			<td>15 min/RT</td>
		</tr>
		<tr>
			<td>3</td>
			<td>95% alcohol</td>
			<td>15 min/RT</td>
		</tr>
		<tr>
			<td>4</td>
			<td>100% alcohol</td>
			<td>15 min/RT</td>
		</tr>
		<tr>
			<td rowspan="4">Clearing</td>
			<td>1</td>
			<td>Xylene</td>
			<td>60 min/4 &#176;C</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Xylene</td>
			<td>10 min/RT</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Xylene</td>
			<td>10 min/RT</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Xylene</td>
			<td>10 min/RT</td>
		</tr>
		<tr>
			<td>*RT, room temperature</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Tissue processing procedure.

Watch this Scientific Journal Video about Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks at JoVE.com

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

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Cancer Research

11.4K Views.  National Cancer Center. Currently, immunofluorescent staining on fixed cells is the method of choice for determination of protein expression levels when morphological information is also necessary. This protocol presented herein provides for an alternative method of immunocytochemistry on paraffin-embedded cell blocks.

Video: Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 &#181;m and 300 &#181;m diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 &#181;m.

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...