Image analysis to quantify histological and immunofluorescent staining of <i>ex vivo</i> skin and skin cell cultures

McMullen, Roger L.; Bauza, E.; Gondran, C.; Oberto, G.; Domloge, N.; Farra, Claude Dal; Moore, David J.

doi:10.1111/j.1468-2494.2010.00541.x

Cited by 15 publications

(3 citation statements)

References 10 publications

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“…For assessment of the collagen density, the area of blue-colored collagen was measured as a percentage of the whole area of the image of the MT-stained slide [32]. For evaluation of TGF-β and fibroblasts, the IF-stained image was observed with a fluorescence microscope (Imager A1; Carl Zeiss, Oberkochen, Germany), and the cells with double positive signals for DAPI and a corresponding antibody were semiquantitatively analyzed as follows: 0; None, 1; Mild, 2; Moderate, and 3; Severe [33].…”

Section: Methodsmentioning

confidence: 99%

Silicone Implant Coated with Tranilast-Loaded Polymer in a Pattern for Fibrosis Suppression

et al. 2019

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Pathologic fibrosis around silicone implants is problematic, and thus, these implants have been coated with a mixture of a biocompatible polymer and antifibrotic drug for sustained drug release to prevent fibrosis. However, a coating applied over an entire surface would be subject to mechanical instability as the implant would be severely crumpled for implant insertion. Therefore, in this work, we proposed localized, patterned coating dots, each composed of poly(lactic-co-glycolic acid) (PLGA) and tranilast, to be applied on the surface of silicone implants. The drug loaded in the pattern-coated implant herein was well retained after a cyclic tensile test. Due to the presence of PLGA in each coating dot, the tranilast could be released in a sustained manner for more than 14 days. When implanted in a subcutaneous pocket in living rats for 12 weeks, compared with the intact implant, the pattern-coated implant showed a decreased capsule thickness and collagen density, as well as less transforming growth factor-β (TGF-β) expression and fewer fibroblasts; importantly, these changes were similar between the surfaces with and without the coating dots. Therefore, we conclude that the pattern-coating strategy proposed in this study can still effectively prevent fibrosis by maintaining the physical stability of the coatings.

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Section: Methodsmentioning

confidence: 99%

Silicone Implant Coated with Tranilast-Loaded Polymer in a Pattern for Fibrosis Suppression

et al. 2019

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“…Analysis of inflammatory cytokine infiltration and epidermal thickness were conducted with ImageJ (NIH). Immunohistochemistry sections were fixed with 4% paraformaldehyde and stained essentially as previously described . Bound primary antibodies (CD11b, F4/80 or IL‐6Rα) were detected by incubation with Alexa Fluor ® 488 or Alexa Fluor ® 546 conjugated secondary antibody (Invitrogen), followed by counterstaining with DAPI (Vector Labs) and visualized under fluorescence using a Leica DM400B microscope.…”

Section: Methodsmentioning

confidence: 99%

IL6Rα function in myeloid cells modulates the inflammatory response during irritant contact dermatitis

Frempah

Luckett-Chastain

Gallucci

2019

Experimental Dermatology

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Irritant contact dermatitis (ICD) is characterized by epidermal hyperplasia, infiltration of leucocytes into lesional skin and inflammatory cytokine release. The cellular infiltrate during ICD comprises primarily cells of the myeloid lineage. Our group has previously shown that the cytokine IL‐6 confers a protective effect to lesional skin during ICD. How IL‐6Rα function in myeloid cells is involved in the inflammatory response during ICD is, however, unknown. In the present study, utilizing a chemical model of ICD, it is shown that mice with a myeloid‐specific knockout of the IL‐6Rα (IL‐6RαΔmyeloid) display an exaggerated inflammatory response to benzalkonium chloride (BKC) and Jet propellant‐8 (JP8) fuel, two well‐characterized irritants relative to littermate control. Results from immunohistochemical and flow cytometric analyses revealed that IL‐6RαΔmyeloid mouse skin displayed increased epidermal hyperplasia and inflammatory monocyte influx into lesional skin but lower numbers of resident macrophages relative to littermate controls after irritant exposure. Multiplex immunoassay revealed significantly higher levels of pro‐inflammatory cytokines IL‐1α and TNF‐α, but reduced expression of chemokine proteins including CCL2‐5, CCL7, CCL11, CXCL1 and CXCL10 in IL‐6RαΔmyeloid mouse skin relative to littermate control following irritant exposure. These results highlight a previously unknown role of IL‐6Rα function in myeloid cells in modulating the inflammatory response and myeloid population dynamics during ICD.

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“…Despite advances in tissue mass spectrometry and microarrays, standard molecular biology approaches exemplified by western blotting, polymerase chain reaction, immunohistochemistry, and high quality imaging of fluorescently stained samples extracted from tumor biopsies are applied in the clinical setting (McMullen et al, 2010; Bogush et al, 2012; Héctor et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Quantitative Assessment of P-Glycoprotein Expression and Function Using Confocal Image Analysis

et al. 2014

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P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at "proof of concept" level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin-Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose-response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

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Image analysis to quantify histological and immunofluorescent staining of ex vivo skin and skin cell cultures

Cited by 15 publications

References 10 publications

Silicone Implant Coated with Tranilast-Loaded Polymer in a Pattern for Fibrosis Suppression

Silicone Implant Coated with Tranilast-Loaded Polymer in a Pattern for Fibrosis Suppression

IL6Rα function in myeloid cells modulates the inflammatory response during irritant contact dermatitis

Quantitative Assessment of P-Glycoprotein Expression and Function Using Confocal Image Analysis

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