Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis
Por:
Rey-Barroso L, Roldan-Molina M, Frías M, Burgos-Fernández FJ, Isola I, Ruiz-Llobet A, Sarrate E and Vilaseca M
Publicada:
1 dic 2025
Ahead of Print:
1 sep 2025
Resumen:
The combined use of immunolabeling and confocal microscopy offers a robust tool for the comprehensive examination of red blood cells, allowing clear visualization of specific proteins and their roles in the cell. While immunolabeling is a widely employed technique, it presents specific challenges in the study of red blood cells, including difficulties with membrane permeabilization, the appearance of background fluorescence, detection of very faint signals, interference from other blood components, and, notably, poor preservation resulting from an incorrect choice of fixation components. Adding to this, fresh blood samples are very labile and need to be preserved as soon as possible, ideally immediately after extraction. Addressing these challenges requires careful experimental design to ensure assay specificity and sensitivity, often complicated by incomplete or unclear information in the literature. In this study, we have compiled available bibliographic data regarding key aspects of red blood cell immunolabeling and devised a detailed immunostaining protocol to investigate the presence of Hemoglobin F in patients with sickle cell anemia treated with hydroxyurea. This protocol ensures proper preservation of red blood cell characteristics and may find application in immunolabeling with other intracellular antibodies.
Filiaciones:
Rey-Barroso L:
Centre for Sensors, Instruments and Systems Development (CD6), Universitat Politècnica de Catalunya (UPC), Rambla Sant Nebridi 10, Terrassa, Barcelona, 08222, Spain
Roldan-Molina M:
Confocal Microscopy and Cellular Imaging Unit, Genetic and Molecular Medicine Department, Instituto Pediátrico de Enfermedades Raras (IPER), Hospital Sant Joan de Déu, Passeig de Sant Joan de Déu 2, Esplugues de Llobregat, Barcelona, 08950, Spain
Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu, Passeig de Sant Joan de Déu 2, Esplugues de Llobregat, Barcelona, 08950, Spain
:
Confocal Microscopy and Cellular Imaging Unit, Genetic and Molecular Medicine Department, Instituto Pediátrico de Enfermedades Raras (IPER), Hospital Sant Joan de Déu, Passeig de Sant Joan de Déu 2, Esplugues de Llobregat, Barcelona, 08950, Spain
Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu, Passeig de Sant Joan de Déu 2, Esplugues de Llobregat, Barcelona, 08950, Spain
Burgos-Fernández FJ:
Centre for Sensors, Instruments and Systems Development (CD6), Universitat Politècnica de Catalunya (UPC), Rambla Sant Nebridi 10, Terrassa, Barcelona, 08222, Spain
Isola I:
Laboratory of Hematology, Service of Laboratory Diagnosis, Hospital Sant Joan de Déu, Passeig de Sant Joan de Déu 2, Esplugues de Llobregat, Barcelona, 08950, Spain
Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu, Passeig de Sant Joan de Déu 2, Esplugues de Llobregat, Barcelona, 08950, Spain
Ruiz-Llobet A:
Service of Pediatric Hematology, Hospital Sant Joan de Déu, Passeig de Sant Joan de Déu 2, Esplugues de Llobregat, Barcelona, 08950, Spain
Institut de Recerca Sant Joan de Déu (IRSJD), Hospital Sant Joan de Déu, Passeig de Sant Joan de Déu 2, Esplugues de Llobregat, Barcelona, 08950, Spain
Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III (ISCIII), Avenida de Monforte de Lemos 5, Madrid, 28029, Spain
Sarrate E:
Laboratory of Hematology, Service of Laboratory Diagnosis, Hospital Sant Joan de Déu, Passeig de Sant Joan de Déu 2, Esplugues de Llobregat, Barcelona, 08950, Spain
Instituto de Investigación de Enfermedades Raras (IIER), Instituto de Salud Carlos III (ISCIII), Avenida de Monforte de Lemos 5, Madrid, 28029, Spain
Vilaseca M:
Centre for Sensors, Instruments and Systems Development (CD6), Universitat Politècnica de Catalunya (UPC), Rambla Sant Nebridi 10, Terrassa, Barcelona, 08222, Spain
Green Accepted
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