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Samples in biobanks are generally preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature compound (OCT)-embedding and subsequently frozen. Mass spectrometry (MS)-based analysis of these samples is now available via developed protocols, however, the differences in results with respect to preservation methods needs further investigation. Here we use bladder urothelial carcinoma tissue of two different tumor stages (Ta/T1—non-muscle invasive bladder cancer (NMIBC), and T2/T3—muscle invasive bladder cancer (MIBC)) which, upon sampling, were divided and preserved by FFPE and OCT. Samples were parallel processed from the two methods and proteins were analyzed with label-free quantitative MS. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Multivariate analysis indicates that the preservation method is the main source of variation, but also tumors of different stages could be differentiated. Proteins involved in mitochondrial function were overrepresented in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Concordant results for proteins such as HMGCS2 (uniquely quantified in Ta/T1 tumors), and LGALS1, ANXA5 and plastin (upregulated in T2/T3 tumors) were observed in both FFPE and OCT data, which supports the use of MS technology for biobank samples and encourages the further evaluation of these proteins as biomarkers., Open access funding provided by Uppsala University., This work was supported by Magnus Bergvall Foundation (S.B.L, 2017-02330, 2018-02726, 2019-03296), Clas Groschinsky memory foundation (S.B.L., M1603, M1742), and Swedish Foundation for Strategic research (S.B.L., SB16-0039). A.V. would like to acknowledge the Ministry of Science and Innovation for a “Juan de la Cierva” postdoctoral Grant.

The ability to measure stem cell mutations is a powerful tool to quantify in a critical cell population if, and to what extent, a chemical can induce mutations that potentially lead to cancer. The use of an enzymatic assay to quantify stem cell mutations in the X-linked glucose-6-phosphate dehydrogenase gene has been previously reported.1 This method requires the preparation of frozen sections and incubation of the sectioned tissue with a reaction mixture that yields a blue color if the cells produce functional glucose-6-phosphate dehydrogenase (G6PD) enzyme. If not, the cells appear whitish. We have modified the reaction mixture using Optimal Cutting Temperature Compound (OCT) medium in place of polyvinyl alcohol. This facilitates pH measurement, increases solubilization of the G6PD staining components and restricts diffusion of the G6PD enzyme. To demonstrate that a mutation occurred in a stem cell, the entire crypt must lack G6PD enzymatic activity. Only if a stem cell harbors a phenotypic G6PD mutation will all of the progeny in the crypt lack G6PD enzymatic activity. To identify crypts with a stem cell mutation, four consecutive adjacent frozen sections (a level) were cut at 7 µm thicknesses. This approach of making adjacent cuts provides conformation that a crypt was fully mutated since the same mutated crypt will be observed in adjacent sections. Slides with tissue samples that were more than 50 µm apart were prepared to assess a total of >104 crypts per mouse. The mutation frequency is the number of observed mutated (white) crypts ÷ by the number of wild type (blue) crypts in a treatment group.

Although numerous studies have examined global mRNA expression in the whole endometrium, very few studies aimed at characterising the global mRNA profile at specific cellular types within this tissue. In this study, we hypothesised that metabolic status during the postpartum period can affect specifically gene expression of endometrial luminal, glandular epithelial and stromal cells. To achieve this, endometrial biopsies were performed in 22 postpartum cows receiving either a control diet (n = 12, aiming for 35 kg/d of energy-corrected milk, ECM) or a mild restricted diet (n = 10, aiming for 25 kg/d of ECM). The diet affected the metabolic status of the cows as documented in Ntallaris et al. (2017 Theriogenology 90, 276-283). Three endometrial biopsies were collected for each cow between 12 and 15 days post-oestrus (oestrus was synchronized with CIDR and all cows had progesterone concentrations >5 ng mL−1 at time of biopsy). Biopsies were frozen in Optimal Cutting Temperature compound and then 10-µm sections were obtained by using cryostat. Slides were fixed in ethanol and stained using cresol violet. Luminal and glandular epithelial cells as well as stromal cells were harvested by laser capture microdissection using the PixCell II LCM System. Following capture, mRNA was extracted from each cellular types using the Arcturus PicoPure RNA Isolation (Thermo Fisher Scientific, Waltham, MA, USA). A genome-wide transcriptomic profile of endometrial cells from biopsies was generated with RNAseq. Samples were sequenced in 50-length paired. The average number of reads across all samples was near 100 million. Reads were mapped on the UMD3.1 assembly of the Bos taurus genome using Tophat. Quantification of gene expression has been performed using HTSeq with annotations from Ensembl. The comparisons of interest have been performed using DESEqn 2 (R package) with the statistical method proposed by Love et al. (2014 Genome Biol. 15, 550). With an adjusted P-value of 0.05, 8, 571, and 2658 genes were found significantly differentially expressed between animals fed with low or control diets for glandular, stromal, and luminal cells respectively. These results provide novel insights into mechanisms regulating endometrium physiology in postpartum dairy cows. The number of differentially expressed genes according to diet was much higher in luminal epithelial and stromal cells than in glandular cells providing evidence that these cells express differential responses to the metabolic environment to which they are exposed. Therefore, transcriptomic analyses based only on full tissue, as possibly biased by differences in cell numbers of different types could be not fully relevant. We acknowledge the financial support of ‘Prolific’ (EU grant KBBE; 31176) and RMUTSV (Thailand)

Frozen human tissues are necessary for research purposes, but tissue banking methods have not changed for more than a decade. Many institutions use cryovial tubes or plastic molds with an optimal cutting temperature compound. However, these methods are associated with several problems, such as samples sticking to one another and the need for a larger storing space. We established an efficient tissue freezing and storing procedure ("tissue tablet method") applicable to both molecular analysis and frozen tissue microarray. Tissue samples were chopped into tiny fragments and embedded into tablet-shaped frozen optimal cutting temperature compound using our original tissue-freezing plate. These tablets can be sectioned and stored in cryovial tubes. We compared the tissue quality of tablet-shaped samples with that of conventional optimal cutting temperature blocks and found no significant difference between them. Tissue microarray is a key method to utilize tissue-banking specimens. However, most tissue microarrays require the coring out of cylindrically shaped tissues from formalin-fixed, paraffin-embedded tissue blocks. Antigenic changes and mRNA degradation are frequently observed with formalin-fixed, paraffin-embedded samples. Therefore, we have applied tablet-shaped samples to construct frozen tissue microarrays with our original mounting base. Constructed tissue microarray sections showed good morphology without obvious artifact and good immunohistochemistry and in situ hybridization results. These results suggest that the quality of arrayed samples was sufficiently appropriate for research purposes. In conclusion, the tissue tablet method and frozen tissue microarray procedure can save time, provides easy tissue handling and processing, and satisfies the demands of research methodologies and tissue banking.