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 80% confluency. Detach cells using a reagent appropriate for that cell line. 
		NOTE: Ethylenediaminetetraacetic acid (EDTA) is generally preferred over trypsin to reduce the risk of degrading surface proteins. If using trypsin, neutralize the trypsin using fetal bovine serum (FBS) immediately after harvesting the cells.</li>
		<li>For suspension cells, harvest approximately 8 x 107 cells in log-phase of growth.</li>
		<li>Collect the harvested/detached cells by centrifuging for 5 min at 225 x g in a 50 mL conical tube.</li>
		<li>Decant the supernatant and re-suspend the pellet in 10 mL of 1X&#160;phosphate-buffered saline (PBS).Centrifuge for 5 min at 225 x g and decant the supernatant. 
		NOTE: 1X PBS is composed of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 in distilled water; adjust pH to 7.4.</li>
	</ol>
	</li>
	<li>Fix the cells in formalin and pellet them.
	<ol>
		<li>Suspend the cell pellet within the conical tube in 10 mL of 10% neutral buffered formalin (NBF). 
		NOTE: NBF can be prepared by diluting 1 mL of 37% formaldehyde stock solution in 9 mL of 1X PBS. 
		CAUTION: Use caution when handling formalin and formaldehyde. Use a fume hood for steps involving formalin and formaldehyde.</li>
		<li>Incubate the cells on a rocker at 24 rpm at room temperature overnight (about 17 h).</li>
		<li>Prepare a 1% solution of low melting point agarose in PBS (0.01 g of agarose per 1 mL PBS).
		<ol>
			<li>Prepare enough for 500 &#181;L of agarose solution per cell line sample.</li>
			<li>Dissolve the agarose in an 80 &#176;C water bath or heat block, with occasional mixing for 2-5 min. Once dissolved, keep the solution in a 37 &#176;C water bath or heat block to prevent hardening.</li>
		</ol>
		</li>
		<li>Pellet the fixed cells from step 1.2.2 by centrifuging for 5 min at 225 x g. Remove the supernatant and resuspend the cells in 500 &#181;L of 1x PBS.</li>
		<li>Transfer the cells into a 1.5 mL microcentrifuge tube and pellet by centrifuging for 5 min at 290 x g. Aspirate off all of the supernatant.</li>
	</ol>
	</li>
	<li>Cast cells in agarose.
	<ol>
		<li>Add ~500 &#181;L of 1% agarose solution (from step 1.2.3) to each tube containing the cells. Gently, but quickly, pipette mixture up and down using a P-1000 micropipette to mix. 
		NOTE: Cut off the end of the pipette tip to enlarge the aperture and avoid forming bubbles. Keep the working agarose solution in the 37 &#176;C water bath to prevent hardening.</li>
		<li>Allow the agarose-cell solution to harden (5-10 min) at room temperature. Add 1 mL of 10% NBF to each microcentrifuge tube containing a sample and keep at room temperature until paraffin embedding (up to 24 h).</li>
	</ol>
	</li>
	<li>Prepare the agarose plug for paraffin embedding.
	<ol>
		<li>Aspirate off the NBF from the sample.</li>
		<li>Remove the cell plug using a razor blade to cut the microcentrifuge tube, place the plug into&#160;plastic tissue cassette. Store cassettes in 10% NBF at room temperature.</li>
	</ol>
	</li>
	<li>Process the samples overnight either manually or using an automated tissue processor, and embed in paraffin wax using standard histology methods. 
	NOTE: See Fischer et al.19&#160;for an example protocol.</li>
	<li>Using a specialized &#34;tissue arrayer&#34; instrument, harvest duplicate 0.6-mm cores from the paraffin block from each cell line and insert them, in rows, into an empty recipient paraffin block in order to create a cell line TMA. 
	NOTE: Standard methods should be used such as those described in Fedor and De Marzo20.</li>
	<li>Incorporate cores from primary samples representing 2-3 additional tissue types (e.g., tonsil, colon, testes, etc.) as positive or negative controls into the TMA. Choose tissues that are appropriate for the protein of interest.</li>
	<li>Use a microtome to prepare two histological sections, approximately 4 to 6 &#181;m thick, of the cell line TMA. Mount the section on a histology slide, dry it, and deparaffinize (as described in Fedor and De Marzo20).</li>
</ol>
2. Sample staining by Immunofluorescence
<ol>
	<li>Optimize the dilution of primary antibody. 
	NOTE: Standard protocols exist for the optimization of IHC or IF for FFPE tissue sections such as in Kajimura et al.21. A brief overview of the approach is outlined here.
	<ol>
		<li>Prepare 4-5&#160;dilutions of primary antibody to the protein of interest guided by the manufacturer&#39;s instructions.</li>
		<li>Identify a control tissue type from an animal or human source that expresses the protein of interest in morphologically recognizable cell populations. Prepare sections of the tissue and mount them on a slide following standard immunohistochemistry procedure such as in Fedor and De Marzo20. 
		NOTE: The number of sections required is the number of primary antibody dilutions plus one extra.</li>
		<li>Using an automated or manual system for immunohistology, test the primary antibody dilutions from step 2.1.1 each on a slide from step 2.1.2. Omit the application of primary antibody from the extra slide and use it as a negative control.</li>
		<li>During the staining process, stain all slides with 4&#39;,6-diamidino-2-phenylindole (DAPI) as a nuclear counterstain and&#160;a fluorescently tagged secondary antibody. If greater sensitivity is required, use an HRP (horseradish peroxidase)-conjugated secondary antibody and tyramide-based signal amplification system (see Stack et al.13). 
		NOTE: When multiplexing primary antibodies, a different fluorescent label is used for each protein.</li>
		<li>Scan the immunostained slides using an appropriate instrument capable of generating a digital image file using excitation and detection wavelengths appropriate to the fluorophores that were used.</li>
		<li>Use appropriate software to view the digital images and empirically choose the primary antibody dilution that optimizes signal intensity relative to background fluorescence. 
		NOTE:&#160;If desired, this optimization can occur using an HRP-conjugated secondary antibody, 3,3&#8217;-diaminobenzidine (DAB) and brightfield microscopy.</li>
	</ol>
	</li>
	<li>Perform IF staining on the cell line TMA.
	<ol>
		<li>Use an automated or manual system for immunohistology to stain a slide of the cell line TMA with the optimized primary antibody dilution (as determined in step 2.1). Omit the application of primary antibody from the second slide and use it as a negative control.</li>
		<li>During the staining process, stain all slides with 4&#39;,6-diamidino-2-phenylindole (DAPI) as a nuclear counterstain, and with a secondary antibody and tyramide as in step 2.1.4.</li>
		<li>Scan the immunostained slides using an appropriate instrument capable of generating a digital image file using excitation and detection wavelengths appropriate to the fluorophores that were used.</li>
		<li>Use an image analysis software package to identify the cellular compartment of interest (i.e., cytoplasm versus nucleus) and quantify the mean fluorescence intensity (MFI) for each cell line. 
		NOTE: Various software packages can be used for this purpose and many are discussed in Stack et al.13.</li>
	</ol>
	</li>
</ol>
3. Quantitative Immunoblotting of Cell Lines
<ol>
	<li>Prepare lysates of cells.
	<ol>
		<li>Harvest 2 million cells of each cell line, as described in steps 1.1.1 to 1.1.3.</li>
		<li>Spin cells down for 5 min at 650 x g in a 50 mL conical tube. Decant the supernatant.</li>
		<li>Wash cells with 10 mL of ice-cold PBS. Resuspend in 1 mL of ice-cold PBS and transfer to a 1.5 mL microcentrifuge tube. Centrifuge as per step 3.1.2, decant, and leave cells on ice.</li>
		<li>Add about 200 &#181;L of cold radioimmunoprecipitation (RIPA) lysis buffer with protease inhibitors (10 &#181;L of 100X&#160;inhibitors per mL of RIPA lysis buffer) to the cells, vortex and incubate on ice for 15 min. 
		NOTE: The amount of lysis buffer required for effective lysis varies by cell line and can be determined empirically. RIPA buffer is composed of 150 mM NaCl, 5 mM EDTA, 50 mM Tris, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in distilled water.</li>
		<li>To ensure relatively even loading on the immunoblots, quantify the total protein in a 20 &#181;L sample from each lysate using an appropriate method such as the Bradford assay. Add about 40 &#181;L of 6X&#160;Laemmli&#160;lysis buffer to the remainder (approximately 180 &#181;L) of each cell lysate and boil at 100 &#176;C in a heat block or water bath for 5 min. 
		NOTE: 6X Laemmli buffer is composed of 300 mM Tris-HCl (pH 6.8), 4% (w/v) SDS, 60% glycerol, 0.6% (w/v) bromophenol blue, 50 mM dithiothreitol (DTT) in distilled water.</li>
		<li>Store the samples at -20 &#176;C for up to two weeks.</li>
	</ol>
	</li>
	<li>Perform an immunoblot of all cells lines to determine which cell line has the most abundant protein of interest. 
	NOTE: Follow the procedure described in Mahmood, T. and Yang, PC.22, with the following modifications.
	<ol>
		<li>Aliquot cell lysate (prepared in step 3.1) containing 10-50 &#181;g of protein (depending on the abundance of the protein of interest) into microcentrifuge tubes. Add 2.5 &#181;L of 6X&#160;Laemmli buffer and sufficient RIPA lysis buffer to make the volume up to 15 &#181;L.</li>
		<li>Load a 5% stacking and an appropriate concentration resolving (e.g., 10% for proteins between 15-100 kDa) SDS-PAGE gel with a protein ladder and the samples from step 3.2.1. Load 1X Laemmli buffer into empty wells. Run the power supply at appropriate settings, typically 125 V, for 80 min or until the bromophenol blue dye reaches the bottom of the plate.</li>
		<li>Perform a semi-dry protein transfer as described in Wiedemann et al.23. 
		NOTE: For the representative results, a nitrocellulose membrane (which does not need to be soaked in methanol), and cold Bjerrum Schafer-Nielsen (BSN) transfer buffer were used. BSN buffer is composed of 48 mM Tris, 39 mM glycine, 20% methanol in distilled water.</li>
		<li>After transfer, use a razor blade to cut the membrane horizontally to separate the portion containing the protein of interest from an appropriate internal control protein, such as GAPDH.</li>
		<li>Proceed to blocking and antibody incubations using the blocking buffer recommended by the manufacturer of the primary antibodies. 
		NOTE: Optimal primary antibody dilutions can vary dramatically depending on protein abundance and sensitivity. For the representative results below, the antibody to the protein of interest required a 1:1,000 dilution while the GAPDH control required a 1:40,000 dilution. The dilution of secondary antibodies used was 1:3,000.</li>
		<li>After antibody incubations and washing of the membrane, place the membrane strips in a clear plastic sheath such as a sandwich bag.</li>
		<li>Prepare enchanced chemiluminescence (ECL) mixture following the manufacturer&#39;s instructions. Use a P-1000 pipette to cover the membrane with the ECL mixture, close the sheath, and incubate the membrane strips with the mixture in the dark at room temperature for 1-2 min.</li>
		<li>Place the membrane sheath in a digital imaging platform. Use chemiluminescence and colorimetric marker detection to capture various exposures of the membrane. 
		NOTE: Exposure times will vary based on the amount of protein loaded, abundance of target protein, antibody affinity etc. Begin with an automatic exposure (typically a few seconds), and test exposure times above and below by increments of a few seconds.</li>
		<li>Empirically, or using image analysis software, determine which cell line expresses the most target protein.</li>
	</ol>
	</li>
	<li>Find the linear dynamic range of each primary antibody using a serial dilution.
	<ol>
		<li>Perform steps 3.2.1-3.2.8 using a series of serial dilutions from the cell line which expresses the highest concentration of the target protein (identified in step 3.2.9).</li>
		<li>Use image analysis software such as ImageJ to perform densitometry on the exposure images.
		<ol>
			<li>For example, using ImageJ, use the Rectangular Selections tool to select the first lane of the gel to quantify. Go to Analyze| Gels| Select First Lane. Use the mouse to move the resulting rectangle over to the next lane. Go to Analyze| Gels| Select Next Lane. Repeatedly move the rectangle to the next lane and select the lane for the remainder of the lanes.</li>
			<li>Go to Analyze| Gels| Plot Lanes. Use the Straight Line tool to draw lines across the bases of each peak to remove background noise. Use the Wand tool to select each peak, and collect the density of each peak, henceforth referred to as band intensity, from the Results window.</li>
		</ol>
		</li>
		<li>Use the densitometry output to create a scatterplot of the band intensity versus the amount of total protein loaded for each primary antibody. Using a line of best fit and visual inspection, determine the location (intensity range) of the linear dynamic range of each antibody.</li>
		<li>Choose a protein concentration that generates a value on the higher end of the linear range to be the concentration moving forward with all cell lines. 
		NOTE: Since this concentration is below the saturation level in the cell line with the greatest amount of this protein, there should be no danger of over exposing the bands for the other cell lines.</li>
	</ol>
	</li>
	<li>Perform an immunoblot using the protein concentration chosen in step 3.3.4 for all cell lines and repeat steps 3.2.1-3.2.8.
	<ol>
		<li>Perform densitometry on the digital scans as in step&#160;3.3.2. Choose the exposures images that yield signals within the linear ranges for each antibody identified in step 3.3.3.</li>
		<li>Using the band intensity signals from the ideal exposures from 3.4.1, calculate the ratio of target protein band intensity to loading control band intensity for each cell line. These ratio values indicate the relative abundance of the target protein of interest.</li>
		<li>Perform a Pearson correlation test (can be done using a statistical software package) to correlate the values obtained from image analysis of the IF staining (step 2.2) to those obtained from immunoblotting (step 3.4.2).</li>
	</ol>
	</li>
</ol>

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W., 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=High+expression+of+Bcl-2+predicts+poor+outcome+in+diffuse+large+B-cell+lymphoma+patients+with+low+international+prognostic+index+receiving+R-CHOP+chemotherapy.">High expression of Bcl-2 predicts poor outcome in diffuse large B-cell lymphoma patients with low international prognostic index receiving R-CHOP chemotherapy.</a> International Journal of Hematology. 103 (2), 210-218 (2016).</li><li>. The Human Protein Atlas BCL2 Available from: <a target="_blank" href="https://www.proteinatlas.org/ENSG00000171791-BCL2/cell#rna">https://www.proteinatlas.org/ENSG00000171791-BCL2/cell#rna</a> (2018)</li><li>Parra, E. R., 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=Validation+of+multiplex+immunofluorescence+panels+using+multispectral+microscopy+for+immune-profiling+of+formalin-fixed+and+paraffin-embedded+human+tumor+tissues.">Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues.</a> Scientific Reports. 7 (1), 13380 (2017).</li><li>Eaton, S. L., 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=A+Guide+to+Modern+Quantitative+Fluorescent+Western+Blotting+with+Troubleshooting+Strategies.">A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies.</a> Journal of Visualized Experiments. (93), e52099 (2014).</li><li>Zellner, M., Babeluk, R., Diestinger, M., Pirchegger, P., Skeledzic, S., Oehler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+Western+blotting+for+quantitation+of+protein+biomarkers+in+clinical+samples.">Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.</a> ELECTROPHORESIS. 29 (17), 3621-3627 (2008).</li><li>Parra, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Platforms+of+Multiplexed+Immunofluorescence+for+Study+of+Paraffin+Tumor+Tissues.">Novel Platforms of Multiplexed Immunofluorescence for Study of Paraffin Tumor Tissues.</a> Journal of Cancer Treatment &amp; Diagnostics. 2 (1), 43-53 (2018).</li><li>Robertson, D., Savage, K., Reis-Filho, J. S., Isacke, 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=Multiple+immunofluorescence+labelling+of+formalin-fixed+paraffin-embedded+(FFPE)+tissue.">Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue.</a> BMC Cell Biology. 9 (1), 13 (2008).</li></ol>

The authors have nothing to disclose.

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

quantitative-immunoblotting-cell-lines-as-standard-to-validate

Research

JoVE Journal

Biology

8.8K Views.  Queen's University. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van kankerpathologie, door biomarkereiwitten te kwantificeren uit routinematig verwerkt biopsiemateriaal. De belangrijkste voordelen van deze techniek in relatie tot conventionele immunohistochemie, zijn dat het objectief is, een breed dynamisch bereik heeft en het mogelijk maakt voor de kwantificering van eiwitten in specifieke celtypen. De aanwezigheid of afwezigheid van bepaalde zogenaamde biomarkereiwitten, ...

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

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Developmental Biology

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemistry

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Cancer Research

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Neuroscience

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 facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

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

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

The Western Blot

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples. Following separation by a technique known as sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, western transfer is used to move proteins from a polyacrylamide gel onto a piece of membrane which traps the proteins in their respective locations. Next, the membranes are probed with antibodies in a process called immunboblotting. Immunoblotting uses antibody-protein and antibody-antibody binding through specific recognition sites, providing the high specificity required for identifying a single protein. The detection of antibodies takes place using reporter systems which includes the use of enzymes. Enzymes can be attached to the end of an antibody and react with substrates to produce changes in color or light. These signals can then be imaged and quantified using a process called densitometry.
This video-article presents an overview of the western blot technique by describing western transfer, the use of antibody detection, and image analysis. The steps involved with western transfer such as the assembly of the transfer sandwich and transfer conditions are discussed in detail as well as the theory behind antibody binding and detection of those antibodies. The broad applications of this technique are described through several examples including the detection of protein-protein interactions and identification of individual proteins within protein complexes.

Western Blotting: Western Transfer, Antibody Detection, and Image Analysis

Western Blotting is used to identify the presence of specific proteins in electrophoretically separated samples.  Following separation by a technique ...

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

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