primary human kidney fibroblast cells  (Innoprot Inc)

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: Representative immunohistochemical micrographs of FFPE and frozen human kidney tissue and human kidney fibroblast (HKF) cells showing peroxidasin (PXDN) expression using two distinct antibody staining methods. Brightfield and corresponding fluorescence images of 4′,6-diamidino-2-phenylindole (DAPI) and PXDN labeling using abx101906 and abx240259 in the FFPE kidney tissue [ a1 and a2 , respectively]. Brightfield images of FFPE kidney tissue stained with abx101906 followed by quenching of endogenous peroxidase activity and incubated with the Lab Vision™ UltraVision™ Large Volume Detection System: anti-Polyvalent, HRP (horseradish peroxidase) (Thermo Fisher Scientific Anatomical Pathology, Cheshire, UK) IHC staining kit and counterstained with hematoxylin (Leica Biosystems, Richmond, IL, USA) [ a3 ]. Fluorescence images of DAPI and PXDN labeling using abx101906 and abx240259 in paraformaldehyde-fixated HKF [ b1 and b2 , respectively] and methanol-fixated HKF stained with abx101906 [ b3 ]. Unstained brightfield and corresponding fluorescence images of DAPI and PXDN labeling using abx101906 in frozen human kidney tissue are displayed in [ c1 ] and [ c2 ], respectively. Details of the histology and staining methods are provided in Materials and Methods. The secondary antibodies used for the immunofluorescence staining were Donkey Anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 647 (Invitrogen, Thermo Fisher Scientific; Waltham, MA, USA; catalog # A32795). All images from human kidney tissue were acquired in a NanoZoomer S60 Digital Slide Scanner (Hamamatsu Photonics; Hamamatsu, Japan). Scale bars: [ a ] and [ c ] 100 μm; [ b ] 20 μm. Color codes depicted in images—bottom left

Article Snippet: Primary HKF cells (Innoprot; Derio, Spain; catalog #P10666) were cultured under standard conditions until reaching approximately 80% confluence.

Techniques: Immunohistochemical staining, Expressing, Staining, Fluorescence, Labeling, Activity Assay, Incubation, Immunohistochemistry, Immunofluorescence

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: Primary HKF cells (Innoprot; Derio, Spain; catalog #P10666) were cultured under standard conditions until reaching approximately 80% confluence.

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