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

Summary

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.

Abstract

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.

Introduction

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 multiplexing^2,3,4. 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 use^5,6,7. Alternative techniques to quantify protein in tissue samples include immunoblotting⁸, mass spectrometry^9,10,11, 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 range^13,14. The superiority, reproducibility, and multiplexing potential of IF applied to FFPE samples has been demonstrated^13,14,15.

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 samples^16,17,18.

Protocol

Approval to use primary human tissue samples was obtained from the Health Sciences and Affiliated Teaching Hospitals Research Ethics Board (HSREB) at Queen's University.

1. Building a Cell-line Tissue Microarray (TMA)

1. Harvest and wash cells.
   NOTE: This protocol has been tested on various established immortalized cell lines (e.g. HeLa, Jurkat, RCH-ACV).
   1. 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.
   2. For suspension cells, harvest approximately 8 x 10⁷ cells in log-phase of growth.
   3. Collect the harvested/detached cells by centrifuging for 5 min at 225 x g in a 50 mL conical tube.
   4. Decant the supernatant and re-suspend the pellet in 10 mL of 1X 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.
2. Fix the cells in formalin and pellet them.
   1. 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.
   2. Incubate the cells on a rocker at 24 rpm at room temperature overnight (about 17 h).
   3. Prepare a 1% solution of low melting point agarose in PBS (0.01 g of agarose per 1 mL PBS).
      1. Prepare enough for 500 µL of agarose solution per cell line sample.
      2. Dissolve the agarose in an 80 °C water bath or heat block, with occasional mixing for 2-5 min. Once dissolved, keep the solution in a 37 °C water bath or heat block to prevent hardening.
   4. 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 µL of 1x PBS.
   5. 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.
3. Cast cells in agarose.
   1. Add ~500 µL 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 °C water bath to prevent hardening.
   2. 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).
4. Prepare the agarose plug for paraffin embedding.
   1. Aspirate off the NBF from the sample.
   2. Remove the cell plug using a razor blade to cut the microcentrifuge tube, place the plug into plastic tissue cassette. Store cassettes in 10% NBF at room temperature.
5. 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 for an example protocol.
6. Using a specialized "tissue arrayer" 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.
7. 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.
8. Use a microtome to prepare two histological sections, approximately 4 to 6 µm thick, of the cell line TMA. Mount the section on a histology slide, dry it, and deparaffinize (as described in Fedor and De Marzo²⁰).

2. Sample staining by Immunofluorescence

1. 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.
   1. Prepare 4-5 dilutions of primary antibody to the protein of interest guided by the manufacturer's instructions.
   2. 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.
   3. 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.
   4. During the staining process, stain all slides with 4',6-diamidino-2-phenylindole (DAPI) as a nuclear counterstain and 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.
   5. 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.
   6. Use appropriate software to view the digital images and empirically choose the primary antibody dilution that optimizes signal intensity relative to background fluorescence.
      NOTE: If desired, this optimization can occur using an HRP-conjugated secondary antibody, 3,3’-diaminobenzidine (DAB) and brightfield microscopy.
2. Perform IF staining on the cell line TMA.
   1. 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.
   2. During the staining process, stain all slides with 4',6-diamidino-2-phenylindole (DAPI) as a nuclear counterstain, and with a secondary antibody and tyramide as in step 2.1.4.
   3. 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.
   4. 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.

3. Quantitative Immunoblotting of Cell Lines

1. Prepare lysates of cells.
   1. Harvest 2 million cells of each cell line, as described in steps 1.1.1 to 1.1.3.
   2. Spin cells down for 5 min at 650 x g in a 50 mL conical tube. Decant the supernatant.
   3. 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.
   4. Add about 200 µL of cold radioimmunoprecipitation (RIPA) lysis buffer with protease inhibitors (10 µL of 100X 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.
   5. To ensure relatively even loading on the immunoblots, quantify the total protein in a 20 µL sample from each lysate using an appropriate method such as the Bradford assay. Add about 40 µL of 6X Laemmli lysis buffer to the remainder (approximately 180 µL) of each cell lysate and boil at 100 °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.
   6. Store the samples at -20 °C for up to two weeks.
2. 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.
   1. Aliquot cell lysate (prepared in step 3.1) containing 10-50 µg of protein (depending on the abundance of the protein of interest) into microcentrifuge tubes. Add 2.5 µL of 6X Laemmli buffer and sufficient RIPA lysis buffer to make the volume up to 15 µL.
   2. 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.
   3. 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.
   4. 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.
   5. 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.
   6. After antibody incubations and washing of the membrane, place the membrane strips in a clear plastic sheath such as a sandwich bag.
   7. Prepare enchanced chemiluminescence (ECL) mixture following the manufacturer'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.
   8. 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.
   9. Empirically, or using image analysis software, determine which cell line expresses the most target protein.
3. Find the linear dynamic range of each primary antibody using a serial dilution.
   1. 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).
   2. Use image analysis software such as ImageJ to perform densitometry on the exposure images.
      1. 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.
      2. 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.
   3. 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.
   4. 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.
4. 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.
   1. Perform densitometry on the digital scans as in step 3.3.2. Choose the exposures images that yield signals within the linear ranges for each antibody identified in step 3.3.3.
   2. 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.
   3. 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).

Results

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...

Discussion

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 IHC^2,3, or by the need to homogenize samples, preventing investigation into the sample structure and cell populations, such as with IB and mass spectro...

Materials

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