Tissue stained with goat antibodies against serum albumin (Immunology Consultant Lab; 1:500) and imaged by a donkey anti-goat antibody conjugated with AlexaFluor-647 (Jackson Immuno, 1:500). Floating sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted on slides. After tissue immunofluorescence was scanned by an Olympus VS120 slide-scanner, serum albumin immunofluorescence was quantified with OlyVIA analysis software (Olympus Life Sciences). Briefly, the areas of the ipsi- and contra-lateral hemisphere was traced in serial section at the TBI lesion epicenter and 180 μm rostral and caudal serial sections. Next, albumin immunofluorescence was set to a minimum intensity-threshold, which remained consistent across all animals to ensure the data was unbiased for each mask-area quantification.
Olyvia analysis software
Manufactured by Olympus
OlyVIA is a software application developed by Olympus for image analysis and processing. It provides a range of tools for visualizing, measuring, and analyzing digital images acquired from various microscopy and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Vs120 slide scanner, by Olympus (1 mentions)
Alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Hrp conjugated goat anti human igg, by Jackson ImmunoResearch (1 mentions)
Anti human igg fc apc antibody, by BioLegend (1 mentions)
Prism 9, by GraphPad (1 mentions)
Ab109390, by Abcam (1 mentions)
Vs120 virtual slide microscope, by Olympus (1 mentions)
Permount mounting medium, by Thermo Fisher Scientific (1 mentions)
Bond rx autostainer, by Leica (1 mentions)
Cellsens, by Olympus (1 mentions)
3 protocols using olyvia analysis software
1
Quantifying Tissue Albumin After TBI
2
Characterizing Nanobodies Binding Affinity
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The activity of Nbs was determined by using indirect ELISA (iELISA), indirect immunofluorescent assay (IFA), and flow cytometry (FCM). For iELISA, BCMA-mFc or CD47-mFc recombinant proteins were coated overnight at 4℃ in the 96-well Immunoplates, washed, blocked with 5% skimmed milk, and then incubated with a dilution series of humanized nanobodies-hFc (huNbs-hFc) for 1 h at 37℃, and HRP-conjugated goat anti-human IgG (cat# 109-035-088, Jackson) was added and incubated for another hour. After washing, 3,3’,5,5’-tetramethylbenzidine (TMB) soluble reagent (cat# PR1200, Solarbio) was added and then the reaction was stopped with 2M H2SO4. The absorbance at 450 nm was measured using an automatic ELISA plate reader. Finally, the binding activity was evaluated using a four-parameter nonlinear regression curve fit (Graphpad Prism 9.0).
For IFA and FCM identification, the expression levels of BCMA on human MM cell lines were detected using APC anti-human BCMA antibody (cat# 357506, Biolegend). Additionally, Alexa Fluor® 594 rabbit anti-human IgG (cat# 309-585-003, Jackson) or APC anti-human IgG Fc antibody (cat# 410712, Biolegend) were incubated with MM cell lines. The z-stack images were acquired using an Olympus SpinSR10 instrument with 100 × objective lenses and analyzed using Olympus OlyVIA analysis software.
For IFA and FCM identification, the expression levels of BCMA on human MM cell lines were detected using APC anti-human BCMA antibody (cat# 357506, Biolegend). Additionally, Alexa Fluor® 594 rabbit anti-human IgG (cat# 309-585-003, Jackson) or APC anti-human IgG Fc antibody (cat# 410712, Biolegend) were incubated with MM cell lines. The z-stack images were acquired using an Olympus SpinSR10 instrument with 100 × objective lenses and analyzed using Olympus OlyVIA analysis software.
3
Quantitative Analysis of Tau Phosphorylation
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Paraffin‐embedded brain sections of 7‐µm‐thick were immunostained for pTau‐ S396 (1:100; #Ab109390, Abcam, MA) and pTau‐S396/S404 (PHF1; 1:250; Peter Davies) using a Bond Rx autostainer (Leica Biosystems, IL), according to the manufacturer's instructions. One set of sections was also incubated for 4 min in thionin to visualize neurons. [23 (link)] Briefly, slides were batch processed with the following settings: Bake and Dewax, IHC protocol F 60 min, HIER 20 min with ER1. Slides were then transferred into water and dehydrated by 1‐min incubations into baths of 70% ethanol, 95% ethanol, 100% ethanol, and xylene. Slides were then cover slipped using Permount Mounting Medium (Fisher Scientific) and left to dry overnight. All the sections were stained at the same time for either pTau‐S396, PHF1, or thionin using the same antibody cocktail. Slides were scanned using an Olympus VS120 virtual slide microscope at a magnification of 20X. Scanned slide images were analyzed in Olympus cellSens and OlyVIA analysis software.
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