Biotechnic & Histochemistry最新文献

Cultivating cells in 3D is considered a significant advancement in cell culture models, as it better reflects natural cellular environments compared to 2D cultures. However, analytical methods like standard light microscopy are less effective for 3D cultures. In this study, 3D cell cultures were generated using the liquid overlay technique with 10,000, 50,000, 100,000 and 200,000 Normal Human Dermal Fibroblasts, analyzed on days 1, 2, and 3 post-seeding. We quantified the influence of fixation with paraformaldehyde or glutardialdehyde/dehydration on their morphology compared to living 3D cell cultures. They were analyzed by light microscopy, scanning electron microscopy as well as by digital light microscopy (height profile measurement). Over time, the cultures decreased in size, likely due to cell shrinkage and structural reorganization. The size reduction could be mathematically described by an exponential decay function. The proportion of round spheroids versus indented aggregates depended on cell number, culture age, and fixation method. On day 1, cultures seeded with 10,000 cells formed nearly 100% round spheroids, regardless of fixation. Higher cell numbers led to fewer round spheroids, and fixation further reduced their number. This suggests that large cell quantities sediment in layers due to steric hindrance, forming indentations. Since aldehydes are responsible for cross-linking proteins, we hypothesize that this chemical reaction, combined with low stability of the 3D cell cultures, leads to the increased formation of the indented 3D cell aggregates. This is consistent with an overall increase in the number of round spheroids and a decrease of the negative influence of fixation over time. In summary, it is important to consider the number of seeded cells, the incubation time, as well as the possible fixation effects when generating stable spheroids using the liquid overlay technique for down-stream experiments.

In this study, we aimed to investigate the specific role of the acetyl-CoA carboxylase (ACACA) gene in oral squamous cell carcinoma (OSCC). We constructed the human tongue carcinoma cell line SAS with low ACACA expression and evaluated changes in cell proliferation, apoptosis, and ferroptosis. Then, the effect of combined treatment with cisplatin and ferroptosis inducer erastin was measured. To assess the impact of ACACA expression on tumor growth in vivo, we established xenograft models with varying ACACA levels in twelve male BALB/c nude mice. ACACA knockdown significantly reduced the proliferation ability of SAS cells, and increased the number of apoptotic cells. ACACA knockdown also induces ferroptosis, and this effect was enhanced by combined treatment with cisplatin and erastin. In vivo experiments demonstrated lower tumor volume and weight in the ACACA knockdown group than those in the control group. Exploring the combined effect of ACACA knockdown and cisplatin treatment revealed a promising synergistic effect against ferroptosis signaling and downstream signaling pathways in SAS cells and in vivo. These findings suggest that targeting the ACACA gene has the potential to be a novel therapeutic strategy for oral cancer treatment.

Dogs represent the most common domesticated animal outside of the research arena for whom tissue for pathological diagnosis is requested. Currently, there is a paucity of literature describing the cross-reactivity of anti-human antibodies on canine tissue for oncological diagnosis. This study aimed to evaluate the cross-reactivity of commonly used anti-human diagnostic antibodies in veterinary histopathology. One hundred seventy-one various samples from 153 canine tissues were processed to paraffin and tested with 76 fully validated anti-human antibody clones frequently employed in hospital pathology laboratories using immunohistochemistry methods to determine which ones exhibited comparable cross-reactivity and therefore potential use in veterinary pathology laboratories. Some of the anti-human antibodies were excluded from the study due to a lack of comparable canine tissues on which to test. However, almost half of the antibodies demonstrated results similar to those in human tissues and about one quarter showed weaker or less consistent labeling. The antibodies that demonstrated weaker or inconsistent labeling suggest areas where further development and modification could improve their employment on canine specimens. Fully one-third of antibodies tested failed to show labeling congruent with human tissues. The outcomes from this study could facilitate the adoption of established anti-human antibody markers in veterinary histopathology practices.

Testicular torsion, a medical condition contributing to male infertility, results in severe scrotal pain and ischemia. Oxidative stress factors contribute to germ cell death following surgical reperfusion, suggesting postoperative pharmacotherapy could mitigate testicular ischemia/reperfusion (I/R) injury. Ivermectin, a GABA receptor modulator for treating parasitic infections such as onchocerciasis, has demonstrated anti-oxidative stress and anti-apoptotic properties. Therefore, this study aimed to evaluate the efficacy of ivermectin administration after detorsion using a rat model of testicular torsion/detorsion (T/D). This study utilized 40 male Wistar rats weighing 200-250 grams. The studied groups were the sham (no T/D induction), control (T/D and no treatment), and T/D following administration of ivermectin administration and the doses of 1, 2, and 5 mg/kg intraperitoneally. For I/R surgery, both testes were twisted 720 degrees clockwise for 4 h to cause torsion. Subsequently, the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) activity were assessed using ELISA, while the expression of GABA_B receptors and apoptosis were examined using the immunohistochemical method. Histopathological alterations were evaluated with hematoxylin and eosin (H&E) staining to evaluate cell counts and seminiferous tubule diameters. Administration of ivermectin effectively reduced oxidative stress levels, as evidenced by decreased MDA levels and increased SOD activity, compared to the control group (P < 0.01 and P < 0.05, respectively). Ivermectin also modulated the expression of elevated GABA_B receptors and caspase 3 (P < 0.05 and P < 0.001, respectively). Furthermore, histopathological analysis revealed that ivermectin prevented germ cell degeneration, edema, and hemorrhage in the testis (P < 0.05). Ivermectin may be a potential treatment option for protecting against testicular torsion and may have beneficial effects on mitigating germ cell death, oxidative stress, and GABA_B receptor modulation. Further research is needed to explore dosages, long-term impacts, and clinical trials to determine the potential for patients with testicular torsion.

Amyloidosis is caused by the extracellular deposition of amyloid fibrils with a β-pleated sheet structure. Diagnosis typically relies on Congo red or Thioflavine T staining. Recently, DAPI (4',6-Diamidino-2-Phenylindole), which is a common nuclear fluorochrome, has been reported to stain amyloid. DAPI staining is simpler than Congo Red and Thioflavine T staining, but its staining mechanism remains unclear. Thus, this study aimed to investigate the mechanism and specificity of DAPI staining for amyloid and its utility. The staining properties of DAPI and Thioflavine T for amyloid were compared on the basis of their stereochemical similarity. In addition, formic acid-treated amyloid specimens were used to investigate the mechanism by which structural changes affect DAPI binding to amyloid fibrils. DAPI staining was also evaluated in four specimens with different types of amyloid. DAPI and Thioflavine T had similar stereochemistry and staining behavior. The amyloid present in formic acid-treated specimens was negative for DAPI staining, indicating that DAPI may recognize the conformation of amyloid fibrils. DAPI stained positively in specimens deposited with AA, Aβ, AL, and AIAPP. DAPI staining recognizes the β-pleated sheet structure of the amyloid fibril structure, and is a simple and sensitive method for detecting amyloid deposition.

Tartrazine, a coal tar-derived azo color, is utilized in food, drinks, cosmetics, and pharmaceuticals. Its azo group catabolizes in the gut, poisoning the liver. This study investigated the efficacy of melatonin, an endogenous antioxidant from the pineal gland against hepatotoxicity in tartrazine-intoxicated rats. Thirty-two adult male wistar albino rats were allocated into four groups: control group, melatonin group (10 mg/kg), tartrazine-treated group (7.5 mg/kg), and tartrazine + melatonin-treated group (7.5 mg/kg tartrazine + 10 mg/kg melatonin). Doses were taken daily for 4 weeks. Melatonin's influence on hepatotoxicity was assessed by monitoring liver enzyme activity, antioxidant state, apoptotic and inflammatory markers, DNA fragmentation, histological and ultrastructural changes. Rats exposed to tartrazine exhibited elevated liver enzymes, oxidant-antioxidant imbalance, and elevated hepatic inflammatory markers (TNF-α, IL-6). Tartrazine also damaged DNA and induced histological and ultrastructural alterations in liver tissue, as shown by the comet assay. Alpha-fetoprotein (AFP) and proliferating cell nuclear antigen (PCNA) were strongly expressed in immunohistochemistry. In rats, melatonin significantly reduced all tartrazine effects. Conversely, melatonin treatment significantly alleviated all aforementioned effects induced by tartrazine in rats by decreasing liver enzymes, elevating antioxidant enzymes, and reducing hepatic inflammatory markers. Enhanced histological assessment and the ultrastructure of the liver was detected following melatonin use. The use of melatonin may safeguard against tartrazine-induced hepatic DNA damage. In conclusion, the current findings indicate that tartrazine administration has detrimental health effects and deleterious impacts on liver function and structure. Melatonin mitigated tartrazine-induced liver damage via antioxidant, anti-inflammatory, and anti-apoptotic pathways.

Periodic acid-Schiff (PAS) staining is useful for visualizing glycogen and mucus. 7-amino-4-methylcoumarin (AMC), an organic reagent, exhibits blue fluorescent signals upon UV excitation. We recently showed that AMC can be used as a fluorescent substitute for Schiff's reagent in PAS staining. In-resin correlative light-electron microscopy (CLEM) is an excellent technique employing fluorescent and electron microscopy (EM) images obtained from the same resin-embedded ultra-thin sections, and provides a high level of morphological concordance between the fluorescent and EM images. Here we studied whether AMC was useful in detecting PAS-positive cells in in-resin CLEM using human pathological samples. Formalin-fixed, paraffin-embedded sections of human colonic and renal tissues and colonic amoebiasis were used. After staining with AMC, these samples were subjected to EM preparation and embedded in epoxy resin. Semi-thin and ultra-thin sections were prepared, and fluorescent and EM images were obtained. AMC signals were well preserved in colonic goblet cells, renal basement membrane, and amoebic bodies in epoxy resin-embedded sections. Using the CLEM technique, a small number of amoebic bodies were easily detected in an inflammatory background of colonic amoebiasis. In conclusion, we successfully detected PAS-positive cells in in-resin CLEM with AMC. Our method may enhance EM analysis in human pathology.