Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some limitations of IMC are its 1-µm resolution and its time and costs of analysis limiting respectively the detailed histopathological analysis of IMC-produced images and its application to small selected tissue regions of interest (ROI) of one to few square millimeters. Coupling on a single-tissue section, IMC and histopathological analyses could permit a better selection of the ROI for IMC analysis as well as co-analysis of immunophenotyping and histopathological data until the single-cell level. The development of this method is the aim of the present study in which we point to the feasibility of applying the IMC process to tissue sections previously Alcian blue-stained and digitalized before IMC tissue destructive analyses. This method could help to improve the process of IMC in terms of ROI selection, time of analysis, and the confrontation between histopathological and immunophenotypic data of cells.

Lignin provides structural support to plants; however, it reduces their utilization rate. According to our previous studies, selenium (Se) reduces lignin accumulation in alfalfa, but the specific mechanism involved remains unclear. Therefore, at the seedling stage, four root irrigation treatments using 2.5, 50, and 5 μmol/L sodium selenite (S-RI), selenomethionine (SS-RI), Se nanoparticles (SSS-RI), and deionized water (CK-RI) were performed. At the branching stage, four treatments of foliar spraying with the three Se fertilizers described above at a concentration of 0.5 mmol/L (S-FS, SS-FS, and SSS-FS) and deionized water (CK-FS) were administered. The results revealed that all Se treatments chiefly reduced the level of deposition of syringyl (S) lignin in the first internode of alfalfa stems. SS-FS and SSS-FS treatments mainly reduced the deposition of S and guaiacyl (G) lignins in the sixth internode of alfalfa stems, respectively, while S-FS treatment only slightly reduced the deposition of G lignin. S, SS, and SSS-RI treatments reduced the level of deposition of S and G lignins in the sixth internode of alfalfa stems. Se application increased plant height, stem diameter, epidermis (cortex) thickness, primary xylem vessel number (diameter), and pith diameter of alfalfa but decreased primary xylem area and pith parenchyma cell wall thickness of the first internode, and SS(SSS)-FS treatment reduced the mechanical strength of alfalfa stems. Therefore, Se application could decrease lignin accumulation by regulating the organizational structure parameters of alfalfa stems and the deposition pattern of the lignin monomers.

BACKGROUND: Staining tissue samples to visualise cellular detail and tissue structure is at the core of pathology diagnosis, but variations in staining can result in significantly different appearances of the tissue sample. While the human visual system is adept at compensating for stain variation, with the growth of digital imaging in pathology, the impact of this variation can be more profound. Despite the ubiquity of haematoxylin and eosin staining in clinical practice worldwide, objective quantification is not yet available. We propose a method for quantitative haematoxylin and eosin stain assessment to facilitate quality assurance of histopathology staining, enabling truly quantitative quality control and improved standardisation.
METHODS: The stain quantification method comprises conventional microscope slides with a stain-responsive biopolymer film affixed to one side, called stain assessment slides. The stain assessment slides were characterised with haematoxylin and eosin, and implemented in one clinical laboratory to quantify variation levels.
RESULTS: Stain assessment slide stain uptake increased linearly with duration of haematoxylin and eosin staining (r = 0.99), and demonstrated linearly comparable staining to samples of human liver tissue (r values 0.98-0.99). Laboratory implementation of this technique quantified intra- and inter-instrument variation of staining instruments at one point in time and across a five-day period.
CONCLUSIONS: The proposed method has been shown to reliably quantify stain uptake, providing an effective laboratory quality control method for stain variation. This is especially important for whole slide imaging and the future development of artificial intelligence in digital pathology.

The precise biological function and activity of the deoxylulose-5-phosphate reductoisomerase (DXR) gene and its promoter in Osmanthus fragrans var. semperflorens remain unclear, even though OfDXR is known as the crucial enzyme involved in plant terpenoid synthesis. This study aimed to shed light on the role and activity of the OfDXR gene and its promoter in O. fragrans var. semperflorens by employing RACE-PCR and Hi-TAIL-PCR techniques for the cloning of the gene and promoter sequence from the petal tissue. Subsequently, genetic transformation and histochemical staining methods were utilized to analyze their function and activity. The OfDXR gene exhibited a DNA sequence length of 5241 bp, encompassing 12 exons and 11 introns. The corresponding cDNA sequence of the OfDXR gene was 1629 bp, encoding 474 amino acid residues. Expression analysis revealed that the OfDXR gene was predominantly active in the petals during the early full blooming stage. Overexpression of the OfDXR gene in Arabidopsis plants at the primary or full blooming stage led to an augmentation in the total terpenoid content. Furthermore, the promoter sequence of the OfDXR gene spanned a length of 1174 bp and contained conserved regulatory/response elements, demonstrating functional activity. These findings indicate that the OfDXR gene plays a pivotal role in terpenoid synthesis, while its promoter exhibits robust activity.

The β-glucuronidase (GUS) reporter gene system is an important technique with versatile uses in the study of flower development in a broad range of species. Transcriptional and translational GUS fusions are used to characterize gene and protein expression patterns, respectively, during reproductive development. Additionally, GUS reporters can be used to map cis-regulatory elements within promoter sequences and to investigate whether genes are regulated post-transcriptionally. Gene trap/enhancer trap GUS constructs can be used to identify novel genes involved in flower development and marker lines useful in mutant characterization. Flower development studies primarily have used the histochemical assay in which inflorescence tissue from transgenic plants containing GUS reporter genes are stained for GUS activity and examined as whole-mounts or subsequently embedded into wax and examined as tissue sections. In addition, quantitative GUS activity assays can be performed on either floral extracts or intact flowers using a fluorogenic GUS substrate. Another use of GUS reporters is as a screenable marker for plant transformation. A simplified histochemical GUS assay can be used to quickly identify transgenic tissues.

Heavy metal pollution possesses potential hazards to plant, animal and human health, which has become the focus of recent attention. Hence, phytoremediation has been regarded as one of the most important remediation technologies for heavy-metal-contaminated soils. In this research, a dominant mine tailing plant, Macleaya cordata, was used as the experimental material to compare the metal transport and oxidative stress response in its roots under lead (Pb) and zinc (Zn) treatments. The result showed that Pb was mainly accumulated in the roots of M. cordata under the Pb treatment; less than 1% Pb was transported to the parts above. An analysis of the Zn content demonstrated a 39% accumulation in the shoots. The production of reactive oxygen species was detected using the in situ histological staining of roots, which showed that hydrogen peroxide in the root tips was observed to increase with the increase in both Pb and Zn concentrations. No significant superoxide anion changes were noted in the root tips under the Pb treatment. An analysis of the root enzyme activity showed that increase in NADPH oxidase activity can be responsible for the production of superoxide anions, subsequent the inhibition of root growth and decrease in antioxidant enzyme activities in the roots of M. cordata exposed to excess Zn. In total, this research provides evidence that the root of M. cordata has a high antioxidant capacity for Pb stress, so it can accumulate more Pb without oxidative damage. On the other hand, the Zn accumulated in the roots of M. cordata causes oxidative damage to the root tips, which can stimulate more Zn transport to the shoots to reduce the damage to the roots. This result will provide a basis for the application of M. cordata in the phytoremediation of soil polluted by Pb-Zn compounds.

The present study aims to discover novel derivatives as antiapoptotic agents and their protective effects against renal ischemia/reperfusion. Therefore, a series of new thiadiazole analogues 2a-g was designed and synthesized through cyclization of the corresponding opened hydrazinecarbothioamides 1a-g, followed by confirmation of the structure via spectroscopic tools (NMR, IR and mass spectra) and elemental analyses. The antiapoptotic activity showed alongside decreasing of tissue damage induced by I/R in the kidneys of rats using N-acetylcysteine (NAC) as an antiapoptotic reference. Most of the cyclized thiadiazoles are better antiapoptotic agents than their corresponding opened precursors. Particularly, compounds 2c and 2g were the most active antiapoptotic compounds with significant biomarkers. A preliminary mechanistic study was performed through caspase-3 inhibition. Compound 2c was selected along with its corresponding opened precursor 1c. An assay of cytochrome C revealed that there is an attenuation of cytochrome C level of about 5.5-fold, which was better than 1c with a level of 4.1-fold. In caspases-3, 8 and 9 assays, compound 2c showed more potency and selectivity toward caspase-3 and 9 compared with 1c. The renal histopathological investigation indicated normal renal tissue for most of the compounds, especially 2c and 2g, relative to the control. Finally, a molecular docking study was conducted at the caspase-3 active site to suggest possible binding modes.

BACKGROUND: Rosette leaf trichomes of Arabidopsis thaliana have been broadly used to study cell development, cell differentiation and, more recently, cell wall biogenesis. However, trichome-specific biochemical or -omics analyses require a proper separation of trichomes from residual plant tissue. Thus, different strategies were proposed in the past for trichome isolation, which mostly rely on harsh conditions and suffer from low yield, thereby limiting the spectrum of downstream analyses.
RESULTS: To take trichome-leaf separation to the next level, we revised a previously proposed method for isolating A. thaliana trichomes by optimizing the mechanical and biochemical specifications for trichome release. We additionally introduced a density gradient centrifugation step to remove residual plant debris. We found that prolonged, yet mild seedling agitation increases the overall trichome yield by more than 60% compared to the original protocol. We noticed that subsequent density gradient centrifugation further visually enhances trichome purity, which may be advantageous for downstream analyses. Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction validated a substantial enrichment upon purification of trichomes by density gradient centrifugation. Histochemical and biochemical investigation of trichome cell wall composition indicated that unlike the original protocol gentle agitation during trichome release largely preserves trichome integrity. We used enriched and density gradient-purified trichomes for proteomic analysis in comparison to trichome-depleted leaf samples and present a comprehensive reference data set of trichome-resident and -enriched proteins. Collectively we identified 223 proteins that are highly enriched in trichomes as compared to trichome-depleted leaves. We further demonstrate that the procedure can be applied to retrieve diverse glandular and non-glandular trichome types from other plant species.
CONCLUSIONS: We provide an advanced method for the isolation of A. thaliana leaf trichomes that outcompetes previous procedures regarding yield and purity. Due to the large amount of high-quality trichomes our method enabled profound insights into the so far largely unexplored A. thaliana trichome proteome. We anticipate that our protocol will be of use for a variety of downstream analyses, which are expected to shed further light on the biology of leaf trichomes in A. thaliana and possibly other plant species.

Oriented cell divisions are crucial throughout plant development to define the final size and shape of organs and tissues. As most of the tissues in mature roots and stems are derived from vascular tissues, studying cell proliferation in the vascular cell lineage is of great importance. Although perturbations of vascular development are often visible already at the whole plant macroscopic phenotype level, a more detailed characterization of the vascular anatomy, cellular organization, and differentiation status of specific vascular cell types can provide insights into which pathway or developmental program is affected. In particular, defects in the frequency or orientation of cell divisions can be reliably identified from the number of vascular cell files. Here, we provide a detailed description of the different clearing, staining, and imaging techniques that allow precise phenotypic analysis of vascular tissues in different organs of the model plant Arabidopsis thaliana throughout development, including the quantification of cell file numbers, differentiation status of vascular cell types, and expression of reporter genes.

Collagen is one of the foremost components of tissue extracellular matrix (ECM). It provides strength, elasticity and architecture to the tissue enabling it to bear the wear and tear from external factors like physical stress as well as internal stress factors like inflammation or other pathological conditions. During normal pregnancy or pregnancy related pathological conditions like preterm premature rupture of membranes (PPROM), collagen of the fetal membrane undergoes dynamic remodeling defining biochemical properties of the fetal membrane. The protocol in this article describes the histochemical method to stain total collagen by Picrosirius red stain which is a simple, quick and reliable method. This protocol can be used on paraformaldehyde (PFA) and formaldehyde fixed paraffin embedded tissue sections. We further describe the staining and distribution of collagen in different mouse reproductive tissues and also demonstrate how this technique in combination with polarization microscopy is useful to detect the distribution of different subtypes of collagen.