Name: quantitative immunoblotting of cell lines as a standard to validate immunofluorescence for quantifying biomarker proteins in routine tissue samples
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples at JoVE.co

This work was partially funded by the Fredrick Banting and Charles Best Canada Graduate Scholarship (A.M.).

Approval to use primary human tissue samples was obtained from the Health Sciences and Affiliated Teaching Hospitals Research Ethics Board (HSREB) at Queen&#39;s University.
1. Building a Cell-line Tissue Microarray (TMA)
<ol>
	<li>Harvest and wash cells. 
	NOTE: This protocol has been tested on various established immortalized cell lines (e.g. HeLa, Jurkat, RCH-ACV).
	<ol>
		<li>For adherent cells, harvest approximately 1.3 x 107 cells once they reach approximately ...

<ol>
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We have described a method that makes use of quantitative immunoblotting (IB) to demonstrate the utility of immunofluorescence (IF) for ascertaining the relative abundance of a target protein in FFPE tissue samples. Current protein quantification methods are limited by their categorical nature, such as chromogenic IHC2,3, or by the need to homogenize samples, preventing investigation into the sample structure and cell populations, such as with IB and mass spectro...

The need to quantify proteins in formalin-fixed, paraffin-embedded (FFPE) tissue biopsy samples exists in many clinical fields. For example, quantification of biomarker proteins in routine biopsy specimens is used to elucidate prognosis and inform treatment for cancer patients1. However, current methods are typically subjective and lack validation.
Immunohistochemistry (IHC) is used routinely in pathology laboratories and generally depends on a primary antibody directed at the target protein and a secondary antibody conjugated with an enzymatic label such as horseradish peroxidase2. Conventional IHC is sensitive, can make use of minute samples and preserves the morphological integrity of tissue samples thereby permitting assessment of protein expression within its relevant histological context. However, because the chromogenic signal generated by IHC is subtractive, it suffers from a relatively narrow dynamic range and offers limited potential for multiplexing2,3,4. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) preserves morphological integrity. However, this developing technology is associated with modest morphological resolution and requires significant calibration and normalization, impairing its feasibility for routine clinical use5,6,7. Alternative techniques to quantify protein in tissue samples include immunoblotting8, mass spectrometry9,10,11, and enzyme-linked immunosorbent assay (ELISA)12, each of which begins with a homogenized lysate of sample tissue. Primary tissue samples are heterogeneous in that they contain a multitude of cell types. Therefore, techniques that entail homogenizing the samples do not permit quantification of a protein in a particular cell population of interest such as cancer cells.
Like IHC, IF is applicable to small FFPE samples and permits the retention of histological integrity13. However, thanks to the additive nature of fluorescence signals, IF is amenable to the application of multiple primary antibodies and fluorescent labels. Thus, a protein of interest may be relatively quantified within specific cells or cellular compartments (for example, nucleus versus cytoplasm) defined using other antibodies. Fluorescence signals also have the advantage of a greater dynamic range13,14. The superiority, reproducibility, and multiplexing potential of IF applied to FFPE samples has been demonstrated13,14,15.
Herein we describe the use of quantitative immunoblotting using established cell lines as a gold standard to ascertain the quantitative nature of IF coupled with computer-assisted image analysis in determining the relative abundance of a protein of interest in histological sections from FFPE tissue samples. We have applied this method successfully in a multiplex approach to quantify biomarker proteins in clinical biopsy samples16,17,18.

<table border="0" cellpadding="0" cellspacing="0" style="width:675px;" width="673">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">697</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 42</td>
			<td style="width:213px;">Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">JeKo-1</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">CRL-3006</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Jurkat</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">TIB-152</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">RCH-ACV</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 548</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Granta-519</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 342</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">REH</td>
			<td style="width:116px;">DSMZ</td>
			<td style="width:123px;">ACC 22</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Raji</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">CCL-86</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">HeLa</td>
			<td style="width:116px;">ATCC</td>
			<td style="width:123px;">CCL-2</td>
			<td>Cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Trypsin/EDTA solution</td>
			<td style="width:116px;">Invitrogen</td>
			<td style="width:123px;">R001100</td>
			<td>For detaching adhesive cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Fetal bovine serum (FBS)</td>
			<td style="width:116px;">Wisent Inc.</td>
			<td style="width:123px;">81150</td>
			<td>To neutralize trypsin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Neutral Buffered Formalin</td>
			<td style="width:116px;">Protocol</td>
			<td style="width:123px;">245-684</td>
			<td>For fixing cell pellets</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">UltraPure low melting point agarose</td>
			<td style="width:116px;">Invitrogen</td>
			<td style="width:123px;">15517-022</td>
			<td>For casting cell pellets</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Mouse monoclonal anti-human Bcl-2 antibody, clone 124</td>
			<td style="width:116px;">Dako (Agilent)</td>
			<td style="width:123px;">cat#: M088729-2, RRID: 2064429</td>
			<td style="width:213px;">To detect Bcl-2 by immunoflourencence and immunoblot (lot#:&#160;00095786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Ventana Discovery XT</td>
			<td style="width:116px;">Roche</td>
			<td style="width:123px;">-</td>
			<td style="width:213px;">For automation of immunofluorescence staining</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">EnVision+ System- HRP labelled polymer (anti-mouse)</td>
			<td style="width:116px;">Dako (Agilent)</td>
			<td style="width:123px;">K4000</td>
			<td style="width:213px;">For immunofluorescence signal amplification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">EnVision+ System- HRP labelled polymer (anti-rabbit)</td>
			<td style="width:116px;">Dako (Agilent)</td>
			<td style="width:123px;">K4002</td>
			<td style="width:213px;">For immunofluorescence signal amplification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Cyanine 5 tyramide reagent</td>
			<td style="width:116px;">Perkin Elmer</td>
			<td style="width:123px;">NEL745001KT</td>
			<td style="width:213px;">For immunofluorescence signal amplification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Aperio ImageScope</td>
			<td style="width:116px;">Leica Biosystems</td>
			<td style="width:123px;">-</td>
			<td style="width:213px;">To view scanned slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">HALO image analysis software</td>
			<td style="width:116px;">Indica Labs</td>
			<td style="width:123px;">-</td>
			<td style="width:213px;">For quantification of immunofluorescence</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Protease inhibitors (Halt protease inhibitor cocktail, 100X)</td>
			<td style="width:116px;">Thermo Scientific</td>
			<td style="width:123px;">1862209</td>
			<td>To add to RIPA buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">EDT001</td>
			<td>For RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NP-40</td>
			<td style="width:116px;">BDH Limited</td>
			<td style="width:123px;">56009</td>
			<td>For RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Sodium deoxycholate</td>
			<td style="width:116px;">Sigma-Aldrich</td>
			<td style="width:123px;">D6750</td>
			<td>For RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Glycerol</td>
			<td style="width:116px;">FisherBiotech</td>
			<td style="width:123px;">BP229</td>
			<td>For Laemlli buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Bromophenol blue</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">BRO777</td>
			<td>For Laemlli buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dithiothreitol (DTT)</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">161-0611</td>
			<td>For Laemlli buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Bovine serum albumin (BSA)</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">ALB001</td>
			<td>For immunoblot washes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Protein ladder (Precision Plus Protein Dual Color Standards)</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">161-0374</td>
			<td>For running protein gel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Filter paper (Extra thick blot paper)</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">1703969</td>
			<td>For blotting transfer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Nitrocellulose membrane</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">162-0115</td>
			<td>For blotting transfer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Trans-blot SD semi-dry transfer cell</td>
			<td style="width:116px;">Bio-Rad</td>
			<td>1703940</td>
			<td>For semi-dry transfer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Skim milk powder (Nonfat dry milk)</td>
			<td style="width:116px;">Cell Signaling Technology</td>
			<td style="width:123px;">9999S</td>
			<td>For blocking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tween 20</td>
			<td style="width:116px;">BioShop</td>
			<td style="width:123px;">TWN510</td>
			<td>For wash buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">GAPDH rabbit monoclonal antibody</td>
			<td style="width:116px;">Epitomics</td>
			<td style="width:123px;">2251-1</td>
			<td style="width:213px;">Primary antibody of control protein (lot#: YE101901C)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Goat anti-mouse IgG (HRP conjugated) antibody</td>
			<td style="width:116px;">abcam</td>
			<td style="width:123px;">cat#: ab6789, RRID: AB_955439</td>
			<td style="width:213px;">Secondary antibody for immunoblot</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:223px;">Goat anti-rabbit IgG (HRP conjugated) antibody</td>
			<td style="width:116px;">abcam</td>
			<td style="width:123px;">cat#: ab6721, RRID: AB_955447</td>
			<td style="width:213px;">Secondary antibody for immunoblot (lot#: GR3192725-5)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Clarity Western ECL substrates</td>
			<td style="width:116px;">Bio-Rad</td>
			<td style="width:123px;">1705060</td>
			<td style="width:213px;">For immunoblot signal detection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Amersham Imager 600</td>
			<td style="width:116px;">GE Healthcare Life Sciences</td>
			<td style="width:123px;">29083461</td>
			<td>For immunoblot signal detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">ImageJ software</td>
			<td style="width:116px;">Freeware, NIH</td>
			<td style="width:123px;">-</td>
			<td>For densitometry analysis</td>
		</tr>
	</tbody>
</table>

quantitative immunoblotting of cell lines as a standard to validate immunofluorescence for quantifying biomarker proteins in routine tissue samples

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

This protocol was used to confirm the ability of IF to determine the relative quantity of the anti-apoptotic protein Bcl-2 in cell lines made into FFPE tissue blocks. Quantifying Bcl-2 selectively in cancer cells can elucidate oncogenic mechanisms and can be useful in pathological diagnosis and in informing clinical management decisions24. More specifically, Bcl-2 plays a role in proper B-lymphocyte development and its expression is commonly investigated in the con...

Watch this Scientific Journal Video about Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples at JoVE.co

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

We describe the use of quantitative immunoblotting to validate immunofluorescence histology coupled with image analysis as a means of quantifying a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Our results demonstrate the utility of immunofluorescence histology for ascertaining the relative quantity of biomarker proteins in routine biopsy samples.

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...

This method can help answer key questions in the field of cancer pathology, by quantifying biomarker proteins from routinely processed biopsy material. The main advantages of this technique in relation to conventional immunohistochemistry, are that it is objective, has a wide dynamic range, and permits for the quantification of proteins in specific cell types. The presence or absence of certain so-called biomarker proteins, or more particularly, the relative abundance of those proteins in cancer biopsy specimens can be very useful in making a specific pathological diagnosis of specific kinds of cancers.But it can also be useful in predicting the response to cancer treatments, so that's why this technique is relevant both to the diagnosis and the treatment of patients with cancer. This protocol is mainly about how to use protein quantification in immortalized cell lines to validate the quantitative nature of immunofluorescence based assay. But it's important to note that immunofluorescence can easily be incorporated into a multiplexed approach, and applied to primary biopsy samples.Assisting and demonstrating this procedure will be Lee Boudreau, a technician, and Shakeel Virk, the director of operations. Both from the Queen's Laboratory for Molecular Pathology. To begin, centrifuge the previously harvested cells in a 50 milliliter conical tube at 225 Gs for five minutes.Decant the supernatant, and resuspend the cell pellet in 10 milliliters of PBS. Then, centrifuge the resuspended cells at 225 Gs for five minutes. Suspend the cell pellet with 10 milliliters of 10%NBF.Then incubate the cells at room temperature on a rocker at 24 RPMs overnight. After fixing the cells, centrifuge the cells at 225 Gs for five minutes. Then remove the supernatant and resuspend the cells in 500 microliters of PBS.Transfer the cells to a 1.5 milliliter microcentrifuge tube, and pellet the cells via centrifugation. Then, aspirate the supernatant. Add approximately 500 microliters of 1%agarose solution to each tube containing cells.Then, pipette the mixture up and down to mix. After the agarose cell solution hardens, add one milliliter of 10%NBF to the tubes. Later, remove the NBF from the samples.Use a razor blade to remove the cell plug, and place the plug into a plastic tissue cassette. Process samples overnight with an automated tissue processor. Then, embed the samples in paraffin wax, using standard histology methods.Using a tissue arrayer instrument, harvest duplicate 6 millimeter cores from the paraffin block, and insert them into an empty paraffin block. After this, use a microtome to prepare two histological sections of the newly created cell line, TMA. Then mount the sections on a histology slide, and deparaffinize them.First, load the two slides of the cell line TMA into an automated staining system. Stain one side with the optimal primary antibody dilution, leaving the other as a negative control slide. To both slides, apply a nuclear counterstain, and secondary antibody.After the staining process, add the tyramide signal amplification reagents. After this, scan the immunostained slides using the appropriate excitation and detection wavelengths for the fluorophores that were used. Use image analysis software to identify the cellular compartment of interest, whether nucleus or cytoplasm, and quantify the mean fluorescence intensity, or MFI, for each line.To determine which cell line has the greatest abundance of the target protein, aliquot preciously prepared cell lysate into microcentrifuge tubes. Then add 2.5 microliters of 6x laemly buffer, and enough RIPA lysis buffer to bring the total volume to 15 microliters. Next, load an SDS page gel, with a protein ladder, and the previously prepared samples.After this, load laemly buffer into the empty wells. Run the gel until the blue dye reaches the bottom of the plate. After performing a semi-dry protein transfer, use a razor blade to cut the membrane horizontally to separate the protein of interest from the control protein.Perform blocking and antibody incubations according to the manufacturers instructions. Then place the membrane strips in a clear plastic bag. Use a P1000 pipette to cover the membranes with ECL mixture.Then seal the bag, and incubate the membranes at room temperature in the dark for one to two minutes. Next, place the plastic bag containing the membranes in a digital imaging platform. Use chemiluminescence and colorimetric marker detection to capture various exposures of the membrane.Using image analysis software, or visual observation, determine which cell line expresses the most target protein. After immunoblotting a serial dilution of this cell line, open the exposure images using image analysis software. Use the rectangular selections tool to select the first lane of the gel.Then go to analyze, gels, and select first lane. Repeat this process by moving the rectangular selections tool over to the next lane, and go to analyze, gels, and select next lane. Next, go to analyze, gels, and plot lanes.Use the straight line tool to draw lines across the bases of each peak, to remove the background noise. Then use the wand tool to select each peak, and obtain the band intensity of each peak from the results window. Create a scatterplot of band intensity versus the amount of total protein loaded for each primary antibody using the densitometry output.Then, use a line of best fit and visual observation to determine the location of the linear dynamic range of each antibody. Select a protein concentration that yields a band intensity within the linear range, and perform an immunoblot of all cell line lysates with that protein concentration, as demonstrated previously. Next, perform densitometry on the digital scans, selecting exposures that yield signals within the previously identified linear ranges for each antibody.After this, calculate the ratio of target protein band intensity to loading control band intensity for each cell line. Finally, use statistical software to perform a Pearson correlation test between the values obtained from image analysis of the IF staining, and those obtained from immunoblotting. This protocol was used to confirm the ability of IF to determine the relative quantity of the anti-apoptotic protein Bcl-2.Varying dilutions of the primary antibody were tested on human tonsil tissue with an immunohistology stainer. For this experiment, a one to 50 dilution was determined to be the optimal dilution that yielded a strong signal with little background noise. This dilution was used to stain the cell line TMA by immunofluorescence, and image analysis was used to quantify the cytoplasmic Cy5 signal attributed to Bcl-2.Based upon the immunoblot for all the tested cell lines, Granta-519 had the greatest abundance of Bcl-2. An immunoblot of the serial dilution of Granta-519 lysate was then used to find the linear dynamic range of Bcl-2, and control protein GAPDH. The dynamic range for Bcl-2 in this assay ranged from a band intensity of nearly 0, to 7500 units.The dynamic range for GAPDH ranged from 3000 to 6500 units. The immunoblot was repeated with exposures within that linear range. And the ratio of Bcl-2 to GAPDH was calculated for each cell line.A Pearson correlation test demonstrated that the intensity ratios from immunoblotting were strongly and positively correlated with the intensity readings from quantitative IF.While attempting this procedure, it's important to remember to expose the final membrane for multiple time points, In order to find the optimal exposure where all of the bands are within their respective linear ranges. After validating the quantitative nature of the IF protocol, it can be modified for multiplex IF, in routinely processed tissue samples in order to answer clinical questions such as where a target protein is expressed.

Research

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Biology

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The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

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Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

A Quantitative Fitness Analysis Workflow

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Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

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Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...