Optimization of Histological Processing of Murine Lung Tissue

In a previous murine study, signs of lung tissue shrinkage and alveolar collapse were consistently found in tissues stored in 70% ethanol for more than 3 months. It was hypothesized that extended storage in 70% ethanol could be the cause of these adverse observations. This study was carried out to: i) identify the cause(s) of the artifacts observed, and ii) investigate the impact of the duration of storage in 70% ethanol prior to processing on the tissue quality. Since 4% formaldehyde is the most commonly used fixative, quality of tissue with fixation in 4% formaldehyde was also evaluated and compared with 70% ethanol. A total of 20 female C57BL/6 mice bred under specific pathogen-free conditions were used. At necropsy, whole lungs were instilled with primary fixative for 48 hours. After this, the left lung lobes were stored in 70% ethanol for the following periods, 1, 3, 6, 9 and 12 weeks and used for the evaluation of the impact of this storage on the tissue quality. The right lung lobes were used to assess the quality of tissue after storing in 4% formaldehyde for the same periods of time. Tissue processing and embedding in paraffin wax were performed according to standard protocols for both left and right lung lobes. Step-serial sections (4-µm thin) were then made from the paraffin embedded right and left lung lobes at intervals of 150 µm, stained with Haematoxylin-Eosin and evaluated by the pathologist. Artifacts were observed in almost all lung tissue samples regardless of the length of the storage in either 70% ethanol or 4% formaldehyde. Tissue alterations observed in this study were comparable to the observation noted in the previous study. As physical compression during embedding was suspected to be the cause of the tissue artifacts, 9 left lung lobes were used to investigate the effect of lung tissue compression. Three left lung lobes were compressed and the remaining lung lobes embedded without any compression. Step-serial sections (4-µm thin) were made at intervals of 150 µm, stained with Haematoxylin-Eosin and evaluated by the pathologist. The use of physical compression of lung tissue during the embedding process was confirmed to result in the observed tissue alterations.