Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Summary

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.

Abstract

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.

Introduction

Red blood cells (RBCs) traverse the cardiovascular system for 70 to 140 days, with a mean RBC age of approximately 115 days^1,2. 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 proteins

Protocol

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 desc....

Results

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

Discussion

Recent literature highly suggests that the RBC-NOS protein is of crucial importance for the regulation of RBC deformability^15,22,23, 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.......

Materials

List of materials used in this article

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