Localisation of connective tissue and inhibition of autofluorescence in the human optic nerve and nerve head using a modified picrosirius red technique and confocal microscopy

Brotchie, Daniel; Birch, Michael; Roberts, Neil; Howard, C. Vyvyan; Smith, Valerie; Grierson, Ian

doi:10.1016/s0165-0270(98)00168-x

Cited by 10 publications

(6 citation statements)

References 26 publications

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“…This staining mechanism indicates that Sirius red nonspecifically binds to basic proteins such as histones in the nucleus. In line with prior findings, 11 this study revealed that PMA pretreatment suppressed nonspecific nuclear staining by PSR in liver tissue.…”

Section: Discussionsupporting

confidence: 93%

“…In this study, we used two protocols for PSR staining, including with (PMA(+)PSR) and without PMA pretreatment (PMA(−)PSR). 11,12 These protocols are presented in Table 1. PMA (12 molybdo (VI) phosphoric acid n -hydrate, H 3 (PMo 12 O 40 ) • nH 2 O) and PSR (CI35780) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…1–4,9 PSR staining can be used to quantify collagen fibers in the periportal, perisinusoidal, and pericentral areas in patients with liver fibrosis. However, Brotchie et al 11 observed nonspecific nuclear staining in the connective tissue of the optic nerve using PSR, and this nonspecific staining was prevented by pretreatment with phosphomolybdic acid (PMA). However, no prior study has used PSR staining with PMA pretreatment to evaluate liver fibrosis.…”

Section: Introductionmentioning

confidence: 99%

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Phosphomolybdic Acid Prevents Nonspecific Nuclear Staining by Picrosirius Red but Is Converted to Molybdenum Blue by Blue Light

Hatori

Moriya

Fujimori

et al. 2020

J Histochem Cytochem.

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Picrosirius red (PSR) staining is generally used to evaluate liver fibrosis; however, PSR sometimes causes nonspecific nuclear staining. In this study, we evaluated the ability of phosphomolybdic acid (PMA) pretreatment to prevent nonspecific nuclear staining by PSR. In a manual evaluation of 27 non-tumor samples from patients with hepatocellular carcinoma, nonspecific nuclear staining was observed in 3.7% of PMA-treated specimens, compared with 85.2% of untreated specimens. Conversely, computer-assisted image analysis (CAIA) identified nonspecific nuclear staining in 0% of PMA-treated samples, vs 44.4% of untreated samples. Surprisingly, after mounting, PMA-treated specimens exhibited a blue tinge because of molybdenum blue (MB) production following sunlight exposure or virtual slide scanning. Using UV cut film, MB production induced by sunlight exposure was prevented; however, the film did not prevent MB production during virtual slide scanning. Moreover, only blue light–emitting diode exposure resulted in a blue tinge in PMA solution. Our data indicated that PMA pretreatment is effective for evaluating liver fibrosis using CAIA. Meanwhile, improvements in virtual slide scanning protocols would directly improve the quality of PMA-pretreated specimens subjected to CAIA:

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Section: Discussionsupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Phosphomolybdic Acid Prevents Nonspecific Nuclear Staining by Picrosirius Red but Is Converted to Molybdenum Blue by Blue Light

Hatori

Moriya

Fujimori

et al. 2020

J Histochem Cytochem.

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“…Its veiling activity on autofluorescence without interfering with the surface fluorescent label resembles the effect of counterstains such as Eriochrome Black T (Kittelberger et al 1989) or picrosirius red (Brotchie et al 1999) used for fluorescence control in frozen sections of blood vessels or nerves, respectively.…”

Section: Discussionmentioning

confidence: 99%

Control of Autofluorescence of Archival Formaldehyde-fixed, Paraffin-embedded Tissue in Confocal Laser Scanning Microscopy (CLSM)

Baschong

Suetterlin

Laeng

2001

J Histochem Cytochem.

263

228

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Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.

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“…After dehydration with ethanol for 30 s, sections were coverslipped. In order to specifically detect collagen, picro-sirius red stained sections were observed via fluorescence microscopy Brotchie et al 1999;Dolber and Spach 1993. Sections were visualized at 100× magnification on an Eclipse E800 microscope (Nikon Instruments, Inc., Melville, NY).…”

Section: Fluorescence Microscopy Of Picro-sirius Red Stained Tehv Secmentioning

confidence: 99%

Prediction of extracellular matrix stiffness in engineered heart valve tissues based on nonwoven scaffolds

Engelmayr

Sacks

2007

Biomech Model Mechanobiol

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The in vitro development of tissue engineered heart valves (TEHV) exhibiting appropriate structural and mechanical characteristics remains a significant challenge. An important step yet to be addressed is establishing the relationship between scaffold and extracellular matrix (ECM) mechanical properties. In the present study, a composite beam model accounting for nonwoven scaffold-ECM coupling and the transmural collagen concentration distribution was developed, and utilized to retrospectively estimate the ECM effective stiffness in TEHV specimens incubated under static and cyclic flexure conditions (Engelmayr Jr et~al. in Biomaterials 26(2):175-187 2005). The ECM effective stiffness was expressed as the product of the local collagen concentration and the collagen specific stiffness (i.e., stiffness/concentration), and was related to the overall TEHV effective stiffness via an empirically determined scaffold-ECM coupling parameter and measured transmural collagen concentration distributions. The scaffold-ECM coupling parameter was determined by flexural mechanical testing of polyacrylamide gels (i.e., ECM analogs) of variable stiffness and associated scaffold-polyacrylamide gel composites (i.e., engineered tissue analogs). The transmural collagen concentration distributions were quantified from fluorescence micrographs of picro-sirius red stained TEHV sections. As suggested by a previous structural model of the nonwoven scaffold (Engelmayr Jr and Sacks in J Biomech Eng 128(4):610-622, 2006), nonwoven scaffold-ECM composites did not follow a traditional rule of mixtures. The present study provided further evidence that the primary mode of reinforcement in nonwoven scaffold-ECM composites is an increase in the number fiber-fiber bonds with a concomitant increase in the effective stiffness of the spring-like fiber segments. Simulations of potential ECM deposition scenarios using the current model indicated that the present approach is sensitive to the specific time course of tissue deposition, and is thus very suitable for studies of ECM formation in engineered heart valve tissues.

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Localisation of connective tissue and inhibition of autofluorescence in the human optic nerve and nerve head using a modified picrosirius red technique and confocal microscopy

Cited by 10 publications

References 26 publications

Phosphomolybdic Acid Prevents Nonspecific Nuclear Staining by Picrosirius Red but Is Converted to Molybdenum Blue by Blue Light

Phosphomolybdic Acid Prevents Nonspecific Nuclear Staining by Picrosirius Red but Is Converted to Molybdenum Blue by Blue Light

Control of Autofluorescence of Archival Formaldehyde-fixed, Paraffin-embedded Tissue in Confocal Laser Scanning Microscopy (CLSM)

Prediction of extracellular matrix stiffness in engineered heart valve tissues based on nonwoven scaffolds

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