A subscription to JoVE is required to view this content. Sign in or start your free trial.
Method Article
Capturing dynamic changes in the protein activation of enucleated red blood cells poses methodological challenges, like the preservation of dynamic changes to acute stimuli for later assessment. The presented protocol describes sample preparation and staining techniques that enable preservation and analysis of relevant protein changes and subsequent detection.
Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute alterations in protein activation states. It facilitates the assessment of RBC treatments, characterization of differences in certain disease states, and description of cellular coherencies. The detection of acutely altered protein activation (e.g., through mechanotransduction) requires adequate sample preparation to preserve otherwise temporary protein modifications. The basic principle includes immobilizing the target binding sites of the desired RBC proteins to enable the initial binding of specific primary antibodies. The sample is further processed to guarantee optimal conditions for the binding of the secondary antibody to the corresponding primary antibody. The selection of non-fluorescent secondary antibodies requires additional treatment, including biotin-avidin coupling and the application of 3,3-diaminobenzidine-tetrahydrochloride (DAB) to develop the staining, which needs to be controlled in real-time under a microscope in order to stop the oxidation, and thus staining intensity, on time. For staining intensity detection, images are taken using a standard light microscope. In a modification of this protocol, a fluorescein-conjugated secondary antibody can be applied instead, which has the advantage that no further development step is necessary. This procedure, however, requires a fluorescence objective attached to a microscope for staining detection. Given the semi-quantitative nature of these methods, it is imperative to provide several control stains to account for non-specific antibody reactions and background signals. Here, we present both staining protocols and the corresponding analytical processes to compare and discuss the respective results and advantages of the different staining techniques.
Red blood cells (RBCs) traverse the cardiovascular system for 70 to 140 days, with a mean RBC age of approximately 115 days1,2. Senescent or damaged RBCs are removed from the circulation by erythrophagocytosis, an efficient clearing process driven by macrophages3. The predetermined lifespan of these cells is one consequence of surrendering the cell organelles, including the nucleus, mitochondria, and ribosomes, during differentiation and maturation4. Thus, circulating RBCs are devoid of a translational machinery, precluding the synthesis of new proteins3. It follows that dynamic, post-translational modifications to existing proteins represent the only viable mechanism of acute, biochemical regulation in response to extracellular and intracellular stressors acting on RBCs5.
Mechanical forces appear to be chief extracellular cues that cause the activation or modulation of biochemical pathways within RBCs. The discovery of the mechanosensitive protein, Piezo1, in RBC membranes6 inspired several lines of research investigating mechanically-activated signaling in these cells7. For example, recent advances have shown that the physical properties of RBCs are actively regulated by acute and dynamic changes of proteins8, which includes post-translational phosphorylation and ubiquitination9. Since these normal modifications differ in certain diseases9,10,11, it seems to be of scientific and clinical interest to determine the activation state of RBC proteins, specifically in relation to mechanobiological processes.
The determination of acute changes in RBC protein activation states poses some methodological challenges. For instance, the storage of RBC samples for later analysis requires preservation of the modified RBC proteins, as post-translational modifications are non-durable. Moreover, classic protein-detection methods (e.g., western blotting) are notoriously difficult to standardize in RBCs due to the low abundance of proteins relative to hemoglobin, which accounts for ~98% of the protein content in these cells12. Thus, antibody-based staining of chemically-preserved RBCs has been the method of choice when investigating acute modifications of important RBC proteins, such as the RBC-specific isoform of nitric oxide synthase (RBC-NOS)13,14. RBC-NOS has been shown to enzymatically produce nitric oxide (NO), which seems indispensable for essential RBC properties, including RBC deformability15,16,17. Post-translational modifications of RBC-NOS regulate catalytic enzyme activity, with phosphorylation of the serine 1177 residue being described to increase enzyme activity, while phosphorylation of the residues serine 114 or threonine 495 have been linked with decreased RBC-NOS activity18,19.
Collectively, temporary modifications of RBC proteins contribute to important cellular function, and standardized protocols that enable detection of these modified proteins are of high value. Here, we present two distinct protocols that exploit specific antibodies to facilitate the detection of RBC-NOS protein activation, and discuss recommendations for data analysis and interpretation.
Performance of the described protocols was assessed by measuring the well-reported increase in the phosphorylation of RBC-NOS at the serine 1177 residue in response to mechanical forces reflective of those occurring within the human vasculature (5 Pa).
The protocols described here are in alignment with the Declaration of Helsinki and were approved by the Ethics Committees of the German Sports University Cologne (9/16/2013) and Griffith University (2019/808). Volunteers were screened to ensure the absence of relevant pathologies and provided written informed consent.
1. Staining of RBC proteins using immunohistochemistry protocols
NOTE: A detailed list of the required chemicals and materials is provided in the Table of Materials. The following sections describe the preparation of the required solutions, followed by a detailed description of the immunohistochemistry protocol (Figure 1).
Figure 1: Schematic of the individual steps required for the immunohistochemical and immunofluorescence staining of RBC-NOS at phosphorylation site 1177. A typical workflow of the presented protocols stretching from solution preparation and blood sampling to antibody-based detection and visualization is presented. Please click here to view a larger version of this figure.
Figure 2: Depiction of the fixative process and blood smear generation. (Scheme created with BioRender.com.) Diluted blood samples are chemically fixed in paraformaldehyde, then centrifuged and washed with phosphate-buffered saline. Finally, resuspended blood is smeared onto a glass slide and thermally fixed via hovering over a Bunsen burner flame. Please click here to view a larger version of this figure.
Table 1: Preparation and storage conditions for solutions necessary for immunohistochemical staining. The solution can be prepared prior to the protocol. Please click here to download this Table.
Table 2: Description of antibody solutions for immediate use. Please click here to download this Table.
Table 3: Components and protocol to prepare DAB solution for immediate use. Please click here to download this Table.
2. Fluorescent labelling of RBC proteins
NOTE: The following section outlines an adaptation of the immunohistochemical protocol, developed with the aim of enabling the use of antibodies with fluorescent conjugates (Figure 1).
Blood sample preparation for the immunofluorescence protocol is identical to that described in section 1, so the following section commences from the staining of samples.
The presented protocol, describing methods that facilitate the detection of acute alterations in RBC proteins, was tested on a well-known mechanically sensitive protein alteration: phosphorylation of RBC-NOS at the serine 1177 residue. Whole blood was obtained from healthy volunteers and subsequently split into two separate aliquots. A given blood sample was exposed to mechanical shear stress of physiological magnitude (5 Pa) for 300 s, which was previously shown to elicit RBC-NOS phosphorylation at serine 1177
Recent literature highly suggests that the RBC-NOS protein is of crucial importance for the regulation of RBC deformability15,22,23, which in turn facilitates their passage through narrow capillaries24. Protein activity highly depends on post-translational protein modifications, particularly the phosphorylation of certain residues18. The focus of interest lies in the phosphorylatio...
All authors have disclosed that there are no conflicts of interest.
LK acknowledges the support of an Australian Government Research Training Program Scholarship.
Name | Company | Catalog Number | Comments |
3,3′-Diaminobenzidin -tetrahydrochloride Hydrate | Sigma/Merck | D5637 | DAB |
Ammoniumchloride | Merck /Millipore | 101145 | NH4Cl |
Centrifuge 5427 R | Eppendorf | 5409000010 | |
Coverslips | VWR | 631-0147 | |
di-sodium Hydrogen Phosphate Dihydrate | Merck /Millipore | 106580 | Na2HPO4. 2 H2O |
Disposable transfer pipettes | VWR | 612-6803 | |
Entellan | Merck /Millipore | 107961 | rapid mounting medium for microscopy |
Ethanol denaturated using 1 % methyl ethyl ketone (MEK) | Hofmann | 642 | |
Glucose-Oxidase | Sigma/Merck | G2133 | |
Grease pencil | Dako | S 2002 | |
Horse-radish peroxidase/ExtrAvidin−Peroxidase | Sigma/Merck | E-2886 | HRP |
Hydrochloric acid | Merck /Millipore | 109057 | HCl |
Hydrogen peroxide, 30% | Merck /Millipore | 107203 | H2O2 |
ImageJ Software | Freeware | ||
Laser-assisted optical rotational cell analyser (LORCA) | RR Mechatronics | Ektacytometer instrument used for shearing | |
Methanol | Merck /Millipore | 106009 | |
Microscope slides | VWR | 630-1985 | |
Nickel(II)-sulfate Hexahydrate | Sigma/Merck | N4882 | NiSO4.6H2O |
Normal Goat serum | Agilent/DAKO | X0907 | NGS |
Paraformaldehyde | Merck /Millipore | 818715 | PFA |
Pipettes Eppendorf Reference 2 | VWR | 613-5836/ 613-5839 | |
Rabbit Anti-phospho eNOS Antibody (Ser1177) | Merck/Millipore | 07-428-I | Primary Antibody |
Reaction tubes, 2ml | Eppendorf | 30120094 | |
Secondary Antibody goat anti rabbit | Agilent/DAKO | E0432 | Secondary Antibody |
Skim milk powder | Bio-Rad | 170-6404 | |
Sodium chloride | Merck /Millipore | 106404 | NaCl |
Sodium Dihydrogen Phosphate Monohydrate | Merck /Millipore | 106346 | NaH2PO4.H2O |
Sodium hydroxide, 1 M | Merck /Millipore | 150706 | NaOH |
Tris(hydroxymethyl)-aminomethane | Merck /Millipore | 108382 | Tris |
Trypsin | Sigma/Merck | T7409 | |
Tween20 | Merck /Millipore | 822184 | |
Whatman Glas microfiber filter, quality GF/F | Merck /Millipore | WHA1825047 | |
Xylol | VWR Chemicals | 2,89,73,465 | |
ß-D-Glucose monohydrate | Merck /Millipore | 14431-43-7 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved