Journal: Histochemistry and Cell Biology
Article Title: Validation and performance assessment of a commercial anti-peroxidasin antibody
doi: 10.1007/s00418-025-02453-7
Figure Lengend Snippet: Validation of anti-peroxidasin (PXDN) antibody specificity: Immunofluorescence histochemistry in human kidney fibroblasts (HKF) under PXDN transient gene silencing and overexpression conditions. Primary HKF (Innoprot; Derio, Spain; catalog #P10666) were treated with either Silencer PXDN siRNA (Ambion, Thermo Fisher Scientific; Waltham, MA, USA; catalog #4392420) for PXDN silencing, or mirVana™ miRNA inhibitor hsa-miR-203 (Ambion, Thermo Fisher Scientific; Waltham, MA, USA; catalog #4464084), using Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific; Waltham, MA, USA; catalog #13778075), according to the Lipofectamine™ RNAiMAX Transfection Reagent Thermo Fisher Protocol. Silencer Selected Negative Control #1 siRNA (Ambion, Thermo Fisher Scientific; Waltham, MA, USA; catalog #4390843) and mirVana™ miRNA inhibitor Negative Control (Ambion, Thermo Fisher Scientific; Waltham, MA, USA; catalog #4464076) were respectively used as negative controls for silencing and overexpression. Following incubation for 5 days of two replicate HKF culture plates, one was used for analysis of PXDN gene expression by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and the other for assessing PXDN protein expression by immunofluorescence cytochemistry. For the RT-qPCR assay, total RNA was extracted with the TripleXtractor direct RNA kit (Grisp; Porto, Portugal; catalog #GK23.0100), reverse-transcribed into cDNA using the PrimeScript RT reagent kit (TaKaRa Biotechnology; Shiga, Japan; catalog #RR014A), and the PXDN expression was quantified as its fold increase relative to the 18S ribosomal RNA housekeeping gene, using the 2 −ΔΔCT method. For immunofluorescence cytochemistry, cells were washed twice with PBS, fixed for 20 min with 4% paraformaldehyde, incubated with blocking/permeabilization buffer (10% normal horse serum and 0.1% Triton X-100 in PBS) for 60 min at room temperature, and incubated overnight at 4 °C in the same buffer, with rabbit polyclonal anti-PXDN antibody (Abbexa; Cambridge, UK; catalog # abx101906). Donkey anti-rabbit IgG (H+L) Alexa Fluor™ Plus 647 (Invitrogen, Thermo Fisher Scientific; Waltham, MA, USA; catalog # A32795) was used as secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, at room temperature. Fluorescence images were acquired in a Leica SP5 Confocal microscope (Leica Microsystems) using the 40× oil objective (exemplary micrographs in [ a ]). [ b1 ]. Fluorescence intensity (arbitrary units (UA)) was retrieved in five randomly selected regions per condition, using ImageJ software [ b2 ]. Scale bars: 50 µm. Color codes: DAPI nuclei staining, blue; PXDN staining, green. Legend: CTRL, Control; Si, PXDN silencing; OE, PXDN overexpression
Article Snippet: For RT-qPCR, total RNA was extracted using the TripleXtractor Direct RNA Kit (Grisp, Porto, Portugal; catalog #GK23.0100), and reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (TaKaRa Biotechnology, Shiga, Japan; catalog #RR014A), and PXDN expression levels were quantified relative to the 18S ribosomal RNA housekeeping gene using the 2 −ΔΔCT method.
Techniques: Biomarker Discovery, Immunofluorescence, Over Expression, Transfection, Negative Control, Incubation, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Blocking Assay, Staining, Fluorescence, Microscopy, Software, Control
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![Validation of anti-peroxidasin (PXDN) antibody specificity: Immunofluorescence histochemistry in human kidney fibroblasts (HKF) under PXDN transient gene silencing and overexpression conditions. Primary HKF (Innoprot; Derio, Spain; catalog #P10666) were treated with either Silencer PXDN siRNA (Ambion, Thermo Fisher Scientific; Waltham, MA, USA; catalog #4392420) for PXDN silencing, or mirVana™ miRNA inhibitor hsa-miR-203 (Ambion, Thermo Fisher Scientific; Waltham, MA, USA; catalog #4464084), using Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific; Waltham, MA, USA; catalog #13778075), according to the Lipofectamine™ RNAiMAX Transfection Reagent Thermo Fisher Protocol. Silencer Selected Negative Control #1 siRNA (Ambion, Thermo Fisher Scientific; Waltham, MA, USA; catalog #4390843) and mirVana™ miRNA inhibitor Negative Control (Ambion, Thermo Fisher Scientific; Waltham, MA, USA; catalog #4464076) were respectively used as negative controls for silencing and overexpression. Following incubation for 5 days of two replicate HKF culture plates, one was used for analysis of PXDN gene expression by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and the other for assessing PXDN protein expression by immunofluorescence cytochemistry. For the RT-qPCR assay, total RNA was extracted with the TripleXtractor direct RNA kit (Grisp; Porto, Portugal; catalog #GK23.0100), reverse-transcribed into cDNA using the <t>PrimeScript</t> RT reagent kit (TaKaRa Biotechnology; Shiga, Japan; catalog <t>#RR014A),</t> and the PXDN expression was quantified as its fold increase relative to the 18S ribosomal RNA housekeeping gene, using the 2 −ΔΔCT method. For immunofluorescence cytochemistry, cells were washed twice with PBS, fixed for 20 min with 4% paraformaldehyde, incubated with blocking/permeabilization buffer (10% normal horse serum and 0.1% Triton X-100 in PBS) for 60 min at room temperature, and incubated overnight at 4 °C in the same buffer, with rabbit polyclonal anti-PXDN antibody (Abbexa; Cambridge, UK; catalog # abx101906). Donkey anti-rabbit IgG (H+L) Alexa Fluor™ Plus 647 (Invitrogen, Thermo Fisher Scientific; Waltham, MA, USA; catalog # A32795) was used as secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min, at room temperature. Fluorescence images were acquired in a Leica SP5 Confocal microscope (Leica Microsystems) using the 40× oil objective (exemplary micrographs in [ a ]). [ b1 ]. Fluorescence intensity (arbitrary units (UA)) was retrieved in five randomly selected regions per condition, using ImageJ software [ b2 ]. Scale bars: 50 µm. Color codes: DAPI nuclei staining, blue; PXDN staining, green. Legend: CTRL, Control; Si, PXDN silencing; OE, PXDN overexpression](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3669/pmc12823669/pmc12823669__418_2025_2453_Fig3_HTML.jpg)