scholarly journals Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment

2004 ◽  
Vol 84 (3) ◽  
pp. 300-306 ◽  
Author(s):  
Vladimir K Rait ◽  
Lixin Xu ◽  
Timothy J O'Leary ◽  
Jeffrey T Mason
2004 ◽  
Vol 121 (2) ◽  
pp. 190-199 ◽  
Author(s):  
Seshi R. Sompuram ◽  
Kodela Vani ◽  
Elizabeth Messana ◽  
Steven A. Bogen

2011 ◽  
Vol 23 (6) ◽  
pp. 1212-1216 ◽  
Author(s):  
Meike M. Mostegl ◽  
Barbara Richter ◽  
Nora Dinhopl ◽  
Herbert Weissenböck

Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral ( Porcine circovirus-2 [PCV-2] and Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents ( Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable with the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, C. serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared with the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable with 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol.


Polymers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1403 ◽  
Author(s):  
Lifen Tong ◽  
Xiting Lei ◽  
Guangyao Yang ◽  
Xiaobo Liu

A novel poly(arylene ether nitrile) terminated with hydroxyl groups (PEN–OH) was synthesized successfully. The effects of heat-treatment temperature on the thermal properties, mechanical properties, and dielectric properties of the PEN–OH films were studied in detail. Due to the cross-linking reaction occurring, at high temperature, among the nitrile groups on the side of the PEN–OH main chain to form a structurally stable triazine ring, the structure of materials changes from a linear structure to a bulk structure. Thus, the thermal properties and mechanical properties were improved. In addition, the occurrence of cross-linking reactions can reduce the polar groups in the material, leading to the decrease of dielectric constant. As the heat-treatment temperature increased, the glass-transition temperature increased from 180.6 °C to 203.6 °C, and the dielectric constant decreased from 3.4 to 2.8 at 1 MHz. Proper temperature heat-treatment could improve the tensile strength, as well as the elongation, at the break of the PEN–OH films. Moreover, because of the excellent adhesive property of PEN–OH to copper foil, a double-layer flexible copper clad laminate (FCCL) without any adhesives based on PEN–OH was prepared by a simple hot-press method, which possessed high peel strength with 1.01 N/mm. Therefore, the PEN–OH has potential applications in the electronic field.


2020 ◽  
Author(s):  
Jian-Hua Wang ◽  
Yu-Liang Tang ◽  
Rohit Jain ◽  
Fan Xiao ◽  
Zhou Gong ◽  
...  

AbstractChemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) has become a widely used method for protein structure analysis. Central to this technology are chemical cross-linkers. The most popular cross-linkers are N-hydroxysuccinimide (NHS) esters, which react with protein amino groups relatively slowly over 10 minutes or more while in competition with the hydrolysis reaction of NHS esters. To improve the speed of cross-linking, we developed a new class of amine-selective and non-hydrolyzable di-ortho-phthalaldehyde (DOPA) cross-linkers. DOPA can cross-link proteins in 10 seconds under near physiological conditions, which is 60 times faster than the NHS ester cross-linker DSS. DOPA also works at low pH, low temperature, or in the presence of high concentrations of denaturants such as 8 M urea or 6 M guanidine hydrochloride. Further, DOPA-mediated pulse cross-linking captured the dynamic conformational changes associated with RNase A unfolding. Lastly, DOPA outperformed DSS at capturing weak but specific protein-protein interactions.


1997 ◽  
Vol 3 (S2) ◽  
pp. 161-162
Author(s):  
S.-R. Shi ◽  
B. Chaiwun ◽  
R.J. Cote ◽  
L. Young ◽  
T. Chen ◽  
...  

The standardization of IHC has been a critical issue since 1977. The most significant challenge for the standardization of routine IHC may be the adverse influence of formalin, which is in part due to variable periods of formalin-fixation ranging from 12 hours to days or weeks, resulting in variable intensity of immunostaining for formalin sensitive antigens. The world-wide use of the AR technique for IHC in both clinical and research histopathology has demonstrated that the AR is a breakthrough in pathology. Based on numerous studies of AR-IHC with excellent results for a variety of interesting markers used in diagnostic pathology, the possibility has been raised that AR technique may be useful in the standardization of IHC. The purpose of this study is to demonstrate the possibility of standardization of IHC based on the ‘test battery‘ approach for establishing an optimal protocol of AR in order to achieve a state of ‘maximal retrieval’, showing a equal comparability of AR-IHC staining of archival paraffin-embedded tissues under varying periods of formalin-fixation.


2020 ◽  
Vol 142 (6) ◽  
pp. 2371-2383
Author(s):  
Sebestyén Nagy ◽  
Erika Fekete ◽  
János Móczó ◽  
Krisztina Koczka ◽  
Emília Csiszár

AbstractCellulose nanocrystals (CNCs) were extracted from natural cellulosic fibres such as bleached cotton and flax with a controlled multi-step sulphuric acid hydrolysis. From the aqueous suspensions of CNCs, the biocomposite films were prepared by casting and evaporation, with an amino-aldehyde (AA) compound in a wide concentration range from 0 to 30%. The AA compound (dimethylol dihydroxy ethylene urea) was considered both as a cross-linker of the CNC and as a matrix polymer for the CNC-reinforced composite system. Two series of films were prepared using different polyols such as sorbitol and glycerol as plasticizers to improve tractability. Heat treatment of the films was performed at elevated temperatures ranging from 140 to 200 °C for 10 min. Results clearly proved that besides temperature, the factors affecting the response of CNC-based nanocomposites to heat treatment were the source of cellulose, the type of plasticizer and the amount of cross-linking agent. Films based on flax–CNC and plasticized with glycerol showed a higher increase in yellowness and a more significant decrease in haze than those derived from cotton–CNC and plasticized with sorbitol, respectively. The cross-linking agent (AA) had a moderating effect on the heat-induced changes of properties. Furthermore, thermal gravimetric analysis (TG) of films revealed that thermal stability of the CNC films improved considerably when AA was added and cross-linking occurred. The increase in Tmax was more significant for the flax–CNC films (from about 230 to 290 °C) than for the cotton–CNC ones (from about 250 to 280 °C).


MicroRNA ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 216-222 ◽  
Author(s):  
Agnès Garcias López ◽  
Louise Brogaard ◽  
Peter Mikail Helwag Heegaard ◽  
Susanna Cirera ◽  
Kerstin Skovgaard

Background: MicroRNAs (miRNAs) are short non-coding RNA molecules which regulate gene expression post-transcriptionally and are involved in a multitude of cellular processes. MiRNAs are known to be very stable compared to messenger RNAs (mRNAs), making them excellent candidates as biomarkers for disease. Recently, studies have suggested that miRNA stability in formalin fixed samples might depend on their nucleotide composition. Objective: To explore the stability of a panel of miRNAs isolated from porcine blood and lung tissue after heat and enzyme treatment. Method: Porcine RNA isolated from lung tissue and blood leukocytes was used for this study. RNA samples were exposed to heat treatment and RNAse A digestion. The levels of selected miRNAs were measured by means of qPCR before and after heat and enzyme treatment. Results: Fourteen miRNAs were successfully analysed, and they were found to degrade differently after exposure to heat or RNAse A. MiRNAs with <60% of adenine (A) and uracil (U) in their sequence were found to be more stable. Conclusion: This is the first study showing that different miRNAs isolated from lung tissue display unequal stability after heat treatment, probably based on their nucleotide composition, highlighting the importance of considering the miRNA sequence when investigating their value as biomarkers.


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