In recent years, Correlative Multimodal Imaging (CMI) has become an \"en vogue\" technique and a bit of a buzzword. It entails combining information from different imaging modalities to extract more information from a sample that would otherwise not be possible from each individual technique. The best established CMI technology is correlative light and electron microscopy (CLEM), which applies light and electron microscopy on the exact same sample/structure. In general, it entails the detection of fluorescently tagged proteins or structures by light microscopy and subsequently their relative intracellular localization is determined with nanometer resolution using transmission electron microscopy (TEM). Here, we describe the different steps involved in a \"simple\" CLEM approach. We describe the overall workflow, instrumentation, and basic principles of sample preparation for a CLEM experiment exploiting stable expression of fluorescent proteins.

Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3;1,4)-β-D-glucan (mixed-linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in 2 Canadian varieties with differing MLG content and nutritional value. We confirmed that oat undergoes a nuclear type of endosperm development but with a shorter cellularization phase than barley (Hordeum vulgare). Callose and cellulose were the first polysaccharides to be detected in the early anticlinal cell walls at 11 days postemergence (DPE) of the panicle. Other polysaccharides such as heteromannan and homogalacturonan were deposited early in cellularization around 12 DPE after the first periclinal walls are laid down. In contrast to barley, heteroxylan deposition coincided with completion of cellularization and was detected from 14 DPE but was only detectable after demasking. Notably, MLG was the last polysaccharide to be laid down at 18 DPE within the differentiation phase, rather than during cellularization. In addition, differences in the spatiotemporal patterning of MLG were also observed between the 2 varieties. The lower MLG-containing cultivar AC Morgan (3.5% w/w groats) was marked by the presence of a discontinuous pattern of MLG labeling, while labeling in the same walls in CDC Morrison (5.6% w/w groats) was mostly even and continuous. RNA-sequencing analysis revealed higher transcript levels of multiple MLG biosynthetic cellulose synthase-like F (CSLF) and CSLH genes during grain development in CDC Morrison compared with AC Morgan that likely contributes to the increased abundance of MLG at maturity in CDC Morrison. CDC Morrison was also observed to have smaller endosperm cells with thicker walls than AC Morgan from cellularization onwards, suggesting the processes controlling cell size and shape are established early in development. This study has highlighted that the molecular processes influencing MLG content and deposition are more complex than previously imagined.

Glutathione peroxidase 2 (Gpx-2) is a selenoenzyme with antioxidant capabilities that may play a role in cancer development. Hence, we investigated the immunohistochemical expression of Gpx-2 protein in colon adenocarcinoma samples derived from patients with colon adenocarcinoma who did not receive any form of treatment prior to the surgical procedure. The associations between the immunohistochemical expression of Gpx-2 and clinical parameters were analysed using the Chi2 test and Fisher\'s exact test. A Kaplan-Meier analysis and the log-rank test were used to verify the relationship between the intensity of Gpx-2 expression and the 5-year survival rate of patients. In total, 101 (80.80%) samples had strong Gpx-2 protein expression and 24 (19.20%) samples were characterized with low expression. The high expression of Gpx-2 was correlated with the histological grade of the tumour (p < 0.001), PCNA immunohistochemical expression (p < 0.001), depth of invasion (p = 0.001) and angioinvasion (p < 0.001). We can conclude that high expression of Gpx-2 is correlated with reduced survival of colon adenocarcinoma patients (log-rank, p < 0.001).

The Notch signalling pathway is one of the most conserved and well-characterised pathways involved in cell fate decisions and the development of many diseases, including cancer. Among them, it is worth noting the Notch4 receptor and its clinical application, which may have prognostic value in patients with colon adenocarcinoma. The study was performed on 129 colon adenocarcinomas. Immunohistochemical and fluorescence expression of Notch4 was performed using the Notch4 antibody. The associations between the IHC expression of Notch4 and clinical parameters were analysed using the Chi2 test or Chi2Yatesa test. The Kaplan-Meier analysis and the log-rank test were used to verify the relationship between the intensity of Notch4 expression and the 5-year survival rate of patients. Intracellular localisation of Notch4 was detected by the use of the immunogold labelling method and TEM. 101 (78.29%) samples had strong Notch4 protein expression, and 28 (21.71%) samples were characterised by low expression. The high expression of Notch4 was clearly correlated with the histological grade of the tumour (p < 0.001), PCNA immunohistochemical expression (p < 0.001), depth of invasion (p < 0.001) and angioinvasion (p < 0.001). We can conclude that high expression of Notch4 is correlated with poor prognosis of colon adenocarcinoma patients (log-rank, p < 0.001).

The Apoptotic protease activating factor 1 (Apaf-1) protein, as one of the factors involved in the activation of the mitochondrial apoptotic pathway, plays an important role in cancer biology. Apaf-1 expression in tumour cells has been shown to be downregulated, with significant implications for tumour progression. Hence, we investigated the expression of Apaf-1 protein in the Polish population of patients with colon adenocarcinoma without any therapy prior to radical surgery. Moreover, we assessed the relation between Apaf-1 protein expression and the clinicopathological factors. The prognostic activity of this protein was analyzed in relation to 5-year survival of patients. In order to show the localization of Apaf-1 protein at the cellular level, the immunogold labelling method was used.
The study was conducted using the colon tissue material from patients with histopathologically confirmed colon adenocarcinoma. Immunohistochemical expression of Apaf-1 protein was performed using Apaf-1 antibody at dilution 1:600. The associations between the immunohistochemistry (IHC) expression of Apaf-1 and clinical parameters were analyzed using the Chi2 test and Chi2Yatesa test. Kaplan-Meier analysis and the log-rank test were used to verify the relationship between the intensity of Apaf-1 expression and 5-year survival rate of patients. The results were considered statistically significant when p < 0.05.
Apaf-1 expression was evaluated by immunohistochemical staining in whole tissue sections. Thirty-nine (33.23%) samples had strong Apaf-1 protein expression and 82 (67.77%) samples were characterized by low expression. The high expression of Apaf-1 was clearly correlated with the histological grade of the tumour (p = 0.001), proliferating cell nuclear antigen (PCNA) immunohistochemical expression (p = 0.005), age (p = 0.015), depth of invasion (p < 0.001) and angioinvasion (p < 0.001). The 5-year survival rate was significantly higher in the group of patients with high expression of this protein (log-rank, p < 0.001).
We can conclude that Apaf-1 expression is positively correlated with reduced survival of colon adenocarcinoma patients.

•  The presence of superoxide dismutase (SOD) enzymes and the response of SOD after in vitro induction and decay of a surface bloom are shown in cultures of the cyanobacterium Microcystis aeruginosa. •  The SOD enzymes of surface blooms, early degenerate and completely degenerate cultures were assayed by staining for SOD activity, immunoblotting and immunogold labelling. •  One band of Mn- and three bands of Fe-SOD were detected in cell extracts. During surface bloom formation, Fe-SOD activity increased fivefold compared with that in control cells; no variation was detected in Mn-SOD activity. However, in early degenerate cultures, Fe-SOD activity decreased to that seen in control cultures, while activity disappeared in completely degenerate cultures. Immunogold labelling showed that Fe-SOD was localized in the cytoplasmic and thylakoid membranes of Microcystis. The extent of labelling paralleled the course of Fe-SOD activity with an increase in particles in surface blooming cells. •  The results suggest Fe-SOD increased due to photooxidative stress. However, under prolonged photooxidative stress, high concentrations of active oxygen species could directly, or indirectly, inactivate and degrade Fe-SOD.

Calcium-dependent cytosolic phospholipase A2α (cPLA2α) had been previously found to be overexpressed by aortic valve interstitial cells (AVICs) subjected to in vitro calcific induction. Here, cPLA2α expression was immunohistochemically assayed in porcine aortic valve leaflets (iAVLs) that had undergone accelerated calcification subsequent to 2- to 28-day-long implantation in rat subcutis. A time-dependent increase in cPLA2α-positive AVICs paralleled mineralization progression depending on dramatic cell membrane degeneration with the release of hydroxyapatite-nucleating acidic lipid material, as revealed by immunogold particles decorating organelle membranes in 2d-iAVLs, as well as membrane-derived lipid byproducts in 7d- to 28d-iAVLs. Additional positivity was detected for (i) pro-inflammatory IL-6, mostly exhibited by rat peri-implant cells surrounding 14d- and 28d-iAVLs; (ii) calcium-binding osteopontin, with time-dependent increase and no ossification occurrence; (iii) anti-calcific fetuin-A, mostly restricted to blood plasma within vessels irrorating the connective envelopes of 28d-iAVLs; (iv) early apoptosis marker annexin-V, limited to sporadic AVICs in all iAVLs. No positivity was found for either apoptosis executioner cleaved caspase-3 or autophagy marker MAP1. In conclusion, cPLA2α appears to be a factor characterizing AVL calcification concurrently with a distinct still uncoded cell death form also in an animal model, as well as a putative target for the prevention and treatment of calcific valve diseases.

Immunogold labelling was used to identify the location of the sucrose hydrolytic enzyme sucrose synthase in the N2 -fixing root nodules of white clover. This is the major enzyme of sucrose cleavage in clover nodules and might be involved in the modulation of N2 fixation by controlling the rate of utilization of photosynthetic products. Knowledge of the precise cellular location of this enzyme in relation to the point of delivery (the vascular bundles) and the site of utilization of the catabolic product (malate in the bacteroids) might aid our understanding of the metabolic communication between different cell types of the nodule. Gold particle density was greatest in the cytosol of uninfected cells of the central region and in the layer of cortical cells in direct contact with infected cells. By contrast, relative gold particle density was 2-3 fold lower in the cytosol of infected cells. The distribution of sucrose synthase is discussed in relation to sucrose transport, starch metabolism and the provision of carbon and energy for N2 fixation.

Immunogold labelling studies using polyclonal antibodies to sucrose synthase suggest that this enzyme is exclusively located in the cytoplasm of soybean nodule cells. A significantly greater intensity of labelling was found in the cytosol of uninfected interstitial cells of the central nodule region compared with the cytosol of the infected cells. Labelling was low in the bulk of the cortical cells but was found to be high in the vascular endodermis and in the cortical cells between the bundle and the infected region. The results are discussed in relation to carbohydrate supply and metabolism and in the context of nodule structure.

•  (1 → 3),(1 → 4)-β-Glucans occur only in the cell walls of the family Poaceae (grasses and cereals) and related families, but little is known about their distribution among walls of different cell types or within walls. •  The locations of (1 → 3)- and (1 → 3),(1 → 4)-β-glucans in the walls of the coleoptile, first leaf and root tip of barley (Hordeum vulgare) seedlings were determined using immunogold labelling. •  All the walls were labelled with the (1 → 3),(1 → 4)-β-glucan antibody, except those of the outer root cap cells. Labelling of the primary walls was heavy in the coleoptile and leaf, but light or very light in the root tip. Two types of primary wall labelling occurred: in the coleoptiles (except the walls of the epidermis and two layers of parenchyma under this) and in the leaf, labelling was throughout the walls; in the root tips, labelling was only adjacent to the plasma membrane. Small amounts of labelling occurred with the (1 → 3)-β-glucan antibody, mostly over plasmodesmata. Both antibodies labelled cell plates. •  (1 → 3),(1 → 4)-β-Glucans occur widely in the cell walls of vegetative organs of the barley seedlings, including the walls of meristematic tissues.