Quantitative immunohistochemistry of fluorescence labelled probes using low-cost software

Kirkeby, Svend; Thomsen, Carsten

doi:10.1016/j.jim.2005.04.006

Cited by 67 publications

(42 citation statements)

References 19 publications

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“…To apply this method, we made sequential shots using CoKin (Cokin SAS, Rungis Cedex, France) filters to obtain all the different color spectra. Adobe Photoshop CS4 extended (Adobe Systems Inc, San Jose, CA) was used to elaborate images [32,33]. Choosing the spectrum related to AEC, we converted the image color profile from RGB to CMYK.…”

Section: Methodsmentioning

confidence: 99%

Immunohistochemical expression and distribution of orexin, orphanin and leptin in the major salivary glands of some mammals

Leone

Spatola

Cucco

et al. 2012

Folia Histochem Cytobiol.

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Abstract:The aim of the study was to determine by immunochemistry the expression of leptin, orexin A and orphanin FQ in the major salivary glands (parotid, submandibular and sublingual) of rat, sheep and cow. These peptides, originally synthesized in central nervous system, adipose tissue and peripheral tissues including gastrointestinal tract, play an orexigenic (orphanin and orexin) or anorexigenic (leptin) roles in the intricate neuronal network appointed to the control of nutritional homeostasis. Peptide-specific immunoreactivity was present in the studied salivary glands with various intensities in different species, in the ductal epithelium, sometimes in the acinar epithelium, and in nervous trunks spread in connective tissue stroma. The obtained data show that salivary glands present an unexpected source of orexigenic and anorexigenic peptides which with their autocrine, paracrine, and endocrine mechanisms of action may participate in the control of salivary gland function.

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Section: Methodsmentioning

confidence: 99%

Immunohistochemical expression and distribution of orexin, orphanin and leptin in the major salivary glands of some mammals

Leone

Spatola

Cucco

et al. 2012

Folia Histochem Cytobiol.

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show abstract

“…Cells were probed with Alexa Fluor 488-labeled monoclonal antihuman HSP70BЈ and were mounted using the VECTASH-IELD HardSet TM with 4Ј,6-diamidino-2-phenylindole antifade mounting medium (Vector Laboratories, Burlingame, CA) and visualized using confocal microscopy (Zeiss LSM 510 laserscanning confocal microscope, Carl Zeiss MicroImaging, Inc. (Thornwood, NY)). Fluorescent images were taken from representative fields of two independent experiments using the same gain and exposure settings, and the amount of green fluorescence was quantified using Adobe Photoshop CS2 to reveal the mean green channel intensity of individual cells as previously described (35).…”

Section: Methodsmentioning

confidence: 99%

Heat Shock Protein 70B′ (HSP70B′) Expression and Release in Response to Human Oxidized Low Density Lipoprotein Immune Complexes in Macrophages

Smith

Twal

Soodavar

et al. 2010

Journal of Biological Chemistry

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Heat shock proteins (HSPs) have been implicated in the activation and survival of macrophages. This study examined the role of HSP70B, a poorly characterized member of the HSP70 family, in response to oxidatively modified LDL (oxLDL) and immune complexes prepared with human oxLDL and purified human antibodies to oxLDL (oxLDL-IC) in monocytic and macrophage cell lines. Immunoblot analysis of cell lysates and conditioned medium from U937 cells treated with oxLDL alone revealed an increase in intracellular HSP70B protein levels accompanied by a concomitant increase in HSP70B extracellular levels. Fluorescence immunohistochemistry and confocal microscopy, however, demonstrated that oxLDL-IC stimulated the release of HSP70B, which co-localized with cell-associated oxLDL-IC. In HSP70B-green fluorescent protein-transfected mouse RAW 264.7 cells, oxLDL-IC-induced HSP70B co-localized with membrane-associated oxLDL-IC as well as the lipid moiety of internalized oxLDL-IC. Furthermore, the data demonstrated that HSP70B is involved in cell survival, and this effect could be mediated by sphingosine kinase 1 (SK1) activation. An examination of regularly implicated cytokines revealed a significant relationship between HSP70B and the release of the anti-inflammatory cytokine interleukin-10 (IL-10). Small interfering RNA knockdown of HSP70B resulted in a corresponding decrease in SK1 mRNA levels and SK1 phosphorylation as well as increased release of IL-10. In conclusion, these findings suggest that oxLDL-IC induce the synthesis and release of HSP70B, and once stimulated, HSP70B binds to the cellassociated and internalized lipid moiety of oxLDL-IC. The data also implicate HSP70B in key cellular functions, such as regulation of SK1 activity and release of IL-10, which influence macrophage activation and survival.

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“…Analysis of confocal images was performed using Adobe Photoshop and Image J software. Unsaturated raw confocal images were standardized as was described previously (30), converted to a single-channel mode and quantified using mean gray value and integrated density algorithms. For actin quantifications, confocal sections in XY were adjusted by using such a combination of lenses and a pinhole value that the resulting images would encompass the entire depth of the BB [Ϯ1.5 m from the center of microvillus (MV)].…”

Section: Reagents and Antibodiesmentioning

confidence: 99%

Identification of intestinal ion transport defects in microvillus inclusion disease

Kravtsov

Ahsan

Kumari

et al. 2016

American Journal of Physiology-Gastrointestinal and Liver Physi

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Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl Ϫ ) and sodium (Na ϩ ) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na ϩ ), CFTR (Cl Ϫ ), and SLC26A3 (DRA) (Cl Ϫ /HCO3 Ϫ ) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl Ϫ and Na ϩ stool loss in MVID diarrhea.CFTR; brush border; MVI; Myo5b; NHE3; MVID MICROVILLUS INCLUSION DISEASE (MVID) is a rare but life-threatening disease that affects newborns and children and leads to rapid death from severe secretory diarrhea. MVID clusters in the Middle East and Navajo Indian populations in the US and is associated with consanguinity (41,42,47,51,61). Stool volumes are greater than 125 ml·kg Ϫ1 ·day Ϫ1 with elevated levels of chloride (Cl Ϫ ) and sodium (Na

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Quantitative immunohistochemistry of fluorescence labelled probes using low-cost software

Cited by 67 publications

References 19 publications

Immunohistochemical expression and distribution of orexin, orphanin and leptin in the major salivary glands of some mammals

Immunohistochemical expression and distribution of orexin, orphanin and leptin in the major salivary glands of some mammals

Heat Shock Protein 70B′ (HSP70B′) Expression and Release in Response to Human Oxidized Low Density Lipoprotein Immune Complexes in Macrophages

Identification of intestinal ion transport defects in microvillus inclusion disease

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