Histomorphometric analyses of human adipose tissues using intact, flash-frozen samples
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Histomorphometric analyses of adipose tissue usually require formalin fixation of fresh samples. Our objective was to determine if intact, flash-frozen whole adipose tissue samples stored at − 80 °C could be used for measurements developed for fresh-fixed adipose tissues. Portions of adipose tissue samples were either formalin-fixed immediately upon sampling or flash-frozen and stored at − 80 °C and then formalin-fixed during the thawing process. Mean adipocyte diameter was measured. Immunohistochemistry was performed on additional samples to identify macrophage subtypes (M1, CD14 + and M2, CD206 +) and total (CD68 +) number. All slides were counterstained using haematoxylin and eosin (H&E). Visual inspection of H&E-stained adipose tissue slides performed in a blinded fashion showed little or no sign of cell breakage in 74% of frozen-fixed samples and in 68% of fresh-fixed samples (p > 0.5). There was no difference in the distribution frequencies of adipocyte sizes in fresh-fixed vs. frozen-fixed tissues in both depots (p > 0.9). Mean adipocyte size from frozen-fixed samples correlated significantly and positively with adipocyte size from fresh-fixed samples (r = 0.74, p < 0.0001, for both depots). The quality of staining/immunostaining and appearance of tissue architecture were comparable in fresh-fixed vs. frozen-fixed samples. In conclusion, intact flash-frozen adipose tissue samples stored at − 80 °C can be used to perform techniques conventionally applied to fresh-fixed samples. This approach allows for retrospective studies with frozen human adipose tissue samples.
KeywordsAdipocyte hypertrophy Immunohistochemistry Image analysis Macrophages Frozen tissue
We would like to acknowledge the contribution of gynecologists and nurses at CHU de Quebec-Laval University as well as the collaboration of participants. We also acknowledge the contribution of Johanne Ouellette from the histology platform (CHU de Quebec) and Debra Harteneck from the Endocrine Research Unit, Mayo Clinic.
The study was supported by operating funds from the Canadian Institutes of Health Research (CIHR) to André Tchernof (MOP-64182). Sofia Laforest was funded by the Centre de recherche en endocrinologie moléculaire et génomique humaine (CREMOGH), by the Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec (CRIUCPQ), by the Canadian Institutes of Health Research (CIHR) (Frederick Banting and Charles Best Canada Graduate Scholarships) and by Fonds de la recherche du Québec-Santé (FRQS). Andréanne Michaud was funded by FRQS and by CIHR (Banting Postdoctoral Fellowships). Denis Soulet holds a Junior 2 Career Award from FRQS.
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Conflict of interest
AT is the recipient of research grant support from Johnson & Johnson Medical Companies for studies unrelated to this publication. No author declared conflict of interest.
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