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Peer-reviewedCC BY-NC 4.0Bio-protocol

Microtube-Assisted Paraffin Embedding and Sectioning of Fragile 3D Tumor Spheroids for High-Quality Histology

  • Ramón Cervantes-Rivera1,2,3
  • Sandra Jetsamari Figueroa Ortiz1
  • Atalia Ziret Romero Rosas1,2
  • Adrián Sánchez Orozco2
  • Ma. Antonia Herrera-Vargas4
  • Esperanza Meléndez-Herrera4
  • Manuel López Rodríguez2
  • Alejandra Ochoa-Zarzosa1,2
  • Joel E. López-Meza1,2
4 affiliations
  1. 1Centro Multidisciplinario de Estudios en Biotecnología (CMEB), Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México
  2. 2Facultad de Medicina Veterinaria y Zootecnia, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México
  3. 3Secretaría de Ciencia, Humanidades, Tecnología e Innovación, Ciudad de México, México
  4. 4Instituto de Investigaciones Sobre los Recursos Naturales. Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México
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Abstract

Three-dimensional (3D) spheroid models have become essential in cancer biology, drug screening, and tissue engineering. However, their small size, fragile structure, and tendency to disintegrate during routine histoprocessing present persistent technical challenges. Conventional paraffin embedding often results in tissue fragmentation, loss of spatial orientation, and poor section quality, whereas cryosectioning often compromises cellular morphology. Here, we present a robust, cost-effective protocol for preserving and sectioning fragile 3D spheroids, resulting in high-quality histological sections with intact architecture and excellent cellular detail. The method involves optimized handling and embedding procedures that stabilize spheroids during standard formalin fixation, paraffin infiltration, and microtomy, eliminating mechanical distortion and preserving spherical integrity for consistent sectioning. We demonstrate the successful application of this protocol across different tumor spheroids derived from distinct tissue types, including MDA-MB-231 (breast carcinoma), Caco-2 (colorectal adenocarcinoma), and A549 (lung carcinoma) cell lines, with subsequent compatibility with hematoxylin and eosin (H&E) staining protocols. This protocol is compatible with immunohistochemistry (IHC) and immunofluorescence (IF). In our qualitative comparison with conventional methods, we found that our approach effectively limits sample loss, promotes consistent inter-section reproducibility, and preserves fine structural features, such as necrotic cores, proliferative zones, and extracellular matrix components. This protocol provides a reliable, accessible solution for routine histological analysis of fragile 3D spheroids, facilitating more accurate morphological assessment in translational research settings.

Key features

• Cost-effective agarose-coated plates for reproducible spheroid formation.

• Complete in-tube processing to minimize sample loss and damage.

• Floating tube system for reliable paraffin infiltration without specialized equipment.

• Produces high-quality sections compatible with H&E.

Graphical overview

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Cite

BibTeXRIS
BibTeX
@article{cervantesrivera2026microtubeassisted,
  title = {{Microtube-Assisted Paraffin Embedding and Sectioning of Fragile 3D Tumor Spheroids for High-Quality Histology}},
  author = {Ramón Cervantes-Rivera and Sandra Jetsamari Figueroa Ortiz and Atalia Ziret Romero Rosas and Adrián Sánchez Orozco and Ma. Antonia Herrera-Vargas and Esperanza Meléndez-Herrera and Manuel López Rodríguez and Alejandra Ochoa-Zarzosa and Joel E. López-Meza},
  journal = {Bio-protocol},
  year = {2026},
  month = oct,
  doi = {10.21769/bioprotoc.5850},
  url = {https://doi.org/10.21769/bioprotoc.5850},
  note = {CC BY-NC 4.0},
}
RIS
TY  - JOUR
TI  - Microtube-Assisted Paraffin Embedding and Sectioning of Fragile 3D Tumor Spheroids for High-Quality Histology
AU  - Ramón Cervantes-Rivera
AU  - Sandra Jetsamari Figueroa Ortiz
AU  - Atalia Ziret Romero Rosas
AU  - Adrián Sánchez Orozco
AU  - Ma. Antonia Herrera-Vargas
AU  - Esperanza Meléndez-Herrera
AU  - Manuel López Rodríguez
AU  - Alejandra Ochoa-Zarzosa
AU  - Joel E. López-Meza
PY  - 2026
DA  - 2026/10/20
JO  - Bio-protocol
DO  - 10.21769/bioprotoc.5850
UR  - https://doi.org/10.21769/bioprotoc.5850
AB  - Three-dimensional (3D) spheroid models have become essential in cancer biology, drug screening, and tissue engineering. However, their small size, fragile structure, and tendency to disintegrate during routine histoprocessing present persistent technical challenges. Conventional paraffin embedding often results in tissue fragmentation, loss of spatial orientation, and poor section quality, whereas cryosectioning often compromises cellular morphology. Here, we present a robust, cost-effective protocol for preserving and sectioning fragile 3D spheroids, resulting in high-quality histological sections with intact architecture and excellent cellular detail. The method involves optimized handling and embedding procedures that stabilize spheroids during standard formalin fixation, paraffin infiltration, and microtomy, eliminating mechanical distortion and preserving spherical integrity for consistent sectioning. We demonstrate the successful application of this protocol across different tumor spheroids derived from distinct tissue types, including MDA-MB-231 (breast carcinoma), Caco-2 (colorectal adenocarcinoma), and A549 (lung carcinoma) cell lines, with subsequent compatibility with hematoxylin and eosin (H&E) staining protocols. This protocol is compatible with immunohistochemistry (IHC) and immunofluorescence (IF). In our qualitative comparison with conventional methods, we found that our approach effectively limits sample loss, promotes consistent inter-section reproducibility, and preserves fine structural features, such as necrotic cores, proliferative zones, and extracellular matrix components. This protocol provides a reliable, accessible solution for routine histological analysis of fragile 3D spheroids, facilitating more accurate morphological assessment in translational research settings.  Key features  • Cost-effective agarose-coated plates for reproducible spheroid formation.  • Complete in-tube processing to minimize sample loss and damage.  • Floating tube system for reliable paraffin infiltration without specialized equipment.  • Produces high-quality sections compatible with H&E.  Graphical overview
SN  - 2331-8325
ER  -