Rat livers were fixed in 4% paraformaldehyde, paraffin-embedded, and sliced into 5 μm sections. Endogenous peroxidases were quenched using 0.3% H2O2 solution followed by blocking using 2.5% goat serum. Mouse anti-human mitochondria IgG (1:1000; Millipore) and goat anti-mouse IgG (Vector) were used as primary and secondary antibodies, respectively. Human mitochondria in the rat liver were visualized using a peroxidase detection kit (immPACT NovaRED Peroxidase (HRP) Substrate, Vector).
Immpact novared peroxidase hrp substrate
Manufactured by Vector Laboratories
Sourced in United States
ImmPACT NovaRED Peroxidase (HRP) Substrate is a chromogenic substrate for immunohistochemical detection of horseradish peroxidase (HRP) enzyme labels. It produces a red-brown precipitate at the site of the HRP enzyme activity.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg, by Vector Laboratories (1 mentions)
Gtx100027, by GeneTex (1 mentions)
Ab125031, by Abcam (1 mentions)
Ds fi1 ccd camera, by Nikon (1 mentions)
Dako envision system hrp labelled polymer detection kit, by Agilent Technologies (1 mentions)
Eclipse ci, by Nikon (1 mentions)
Anti pecam 1, by Santa Cruz Biotechnology (1 mentions)
Anti vegf, by Santa Cruz Biotechnology (1 mentions)
Ab955, by Abcam (1 mentions)
Streptavidin biotin blocking kit, by Vector Laboratories (1 mentions)
Sox10, by Santa Cruz Biotechnology (1 mentions)
Pd l1, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit antibodies, by Bio-Rad (1 mentions)
Eukitt, by Merck Group (1 mentions)
Aperio imagescope, by Leica (1 mentions)
8 protocols using immpact novared peroxidase hrp substrate
1
Visualizing Human Mitochondria in Rat Liver
2
Immunohistochemical Analysis of CXCR7 in Lung Cancer
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A total of 47 patients with lung cancer were included in the study. A tissue microarray (TMA) containing 43 formalin fixed paraffin embedded (FFPE) primary lung tumor samples from the Department of Pathology, University Medical Center Groningen (UMCG), and 4 additional FFPE primary lung tumor samples from The First Hospital of Lanzhou University were used to detect CXCR7 protein expression using IHC. The study was performed in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines.
Briefly, 4 μm FFPE sections were deparaffinized using xylene for 10 min. Next, slides were incubated with Tris/HCl (pH = 9) in a microwave. After blocking endogenous peroxidase activity with hydrogen peroxide, slides were incubated with the anti-CXCR7 primary rabbit polyclonal antibody (GTX100027, GeneTex, Irvine, USA) for 1 h at RT. Slides were then incubated with peroxidase-labeled goat anti-rabbit secondary and rabbit anti-goat tertiary antibodies (Dako, Denmark) for 30 min at RT. Visualization was performed using ImmPACT NovaRED Peroxidase (HRP) Substrate (Vector Laboratories, Burlingame, CA, USA) followed by hematoxylin staining. Scoring of the slides was performed by an experienced pulmonary pathologist (WT).
Briefly, 4 μm FFPE sections were deparaffinized using xylene for 10 min. Next, slides were incubated with Tris/HCl (pH = 9) in a microwave. After blocking endogenous peroxidase activity with hydrogen peroxide, slides were incubated with the anti-CXCR7 primary rabbit polyclonal antibody (GTX100027, GeneTex, Irvine, USA) for 1 h at RT. Slides were then incubated with peroxidase-labeled goat anti-rabbit secondary and rabbit anti-goat tertiary antibodies (Dako, Denmark) for 30 min at RT. Visualization was performed using ImmPACT NovaRED Peroxidase (HRP) Substrate (Vector Laboratories, Burlingame, CA, USA) followed by hematoxylin staining. Scoring of the slides was performed by an experienced pulmonary pathologist (WT).
3
Immunohistochemical Analysis of MC1R in Clinical Melanoma
4
Histological and Immunohistochemical Analysis of Wound Healing
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Histology and immunohistochemistry were performed as previously described [22 (link)]. Briefly, wound tissue was fixed with 10% neutral-buffered formalin and paraffin sections (4 μm) stained with Mayer’s haematoxylin and eosin on randomly chosen samples. For immunohistochemistry, sections were incubated with rabbit anti-MMP-2 and anti-MMP-9 and isotyped-matched rabbit IgG (control; 1 µg/mL, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). For immunodetection, a Dako EnVision+ System-HRP labelled polymer detection kit (Dako, Carpinteria, CA, USA) was used with ImmPACT NovaRED peroxidase (HRP) substrate (Vector Laboratories, Burlingame, CA, USA), and counterstained using Mayer’s hematoxylin and Scott’s bluing solution. After mounting, sections were observed under a light microscope (Eclipse Ci; Nikon, Tokyo, Japan) and micro-graphed using a DS-Fi1 CCD camera (Nikon, Tokyo, Japan). All samples were stained in a single assay to exclude between-run variability. The wound gap was measured by drawing a line the width of the wound, and the number of pixels was recorded using ImageJ. The pixels of the line were divided by the pixels of the scale bar as indicated in the figures.
5
Histological and Immunohistochemical Assessment of Tissue
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Samples were harvested from the induced membrane and local fascia (control) during the second stage surgical procedure. Specimens were approximately 10 × 30 mm in size, with 10 × 20 mm of this used for histology and IHC. After harvest, all specimens were rinsed in PBS and fixed in 4% paraformaldehyde for 48 to 72 hours, depending on the thickness of the sample, at 4°C. Tissue samples were decalcified in 10% EDTA for 10 days on a shaker at room temperature, before being embedded in paraffin, and sectioned at 5 μm. The samples were stained with Hematoxylin & Eosin (H&E) and Masson Trichrome to determine tissue morphology. IHC was performed using anti-CD68 antibody (1:100, ab955, Abcam plc, Cambridge, UK), anti-PECAM-1 (1:100, sc-376764) or anti-VEGF (1:200, sc-7269) (Santa Cruz Biotechnology, Inc., Dallas, TX). Antigens were retrieved using Proteinase K and the sections were incubated in primary antibody dilutions overnight at 4°C. The Mouse and Rabbit Specific HRP (ABC) Detection IHC kit (Sigma-Aldrich Co., St. Louis, MO) in combination with the ImmPACT NovaRED Peroxidase (HRP) Substrate (Vector Laboratories, Inc., Burlingame, CA) was used for antibody detection before the sections were counterstained with 10% hematoxylin.
6
Immunofluorescence and Immunohistochemical Staining
7
Immunohistochemical Analysis of Tumor Tissue
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Tumor sections were fixed in 4% paraformaldehyde. The antibodies used were G9a (Cell Signaling Technology), LC3B (Santa Cruz Biotechnology), SOX10 (Santa Cruz Biotechnology), and PD-L1 (Abcam). The universal secondary protocol and the 3,3 0 -Diaminobenzidine (DAB; Biocare Medical) or ImmPact NovaRed Peroxidase (HRP) substrate (Vector Laboratories) were used to detect and amplify the signal. Aperio ImageScope software (ImageScope, RRID: SCR_014311) was used for imaging and quantitation of five nonoverlapping tumor regions and evaluating the number of positive pixels per unit area in each region.
8
Phosphorylated HSL Immunohistochemistry in Rat Liver
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Immunohistochemistry for phosphorylated HSL (anti-pHSL; Supplementary Table S2) was performed on paraffin-embedded rat liver sections as previously described [29] (link). Briefly, after deparaffinization antigen retrieval was performed by microwave irradiation in citrate buffer (10 mmol/L), pH 6.0 and blocking of endogenous peroxidase with 0.3% H 2 O 2 for 30 min. After blocking (1% BSA for 30 min), tissue was incubated with polyclonal-rabbit-phospho-HSL (Ser660) primary antibody (1:50 dilution in 1% BSA) overnight at 4 °C in a humidity chamber. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies (1:50; #170-6515, Bio-Rad) was used as secondary antibody. Slides were stained with ImmPact NovaRED Peroxidase (HRP) substrate (cat# SK-4805, Vector Laboratories, The Netherlands) for 11 min, and hematoxylin was used as a counter nuclear stain (2 min at RT). Finally, slides were dehydrated and mounted with Eukitt®, (Sigma-Aldrich). Slides were scanned using a nanozoomer 2.0 HT digital slide scanner (C9600-12, Hamamatsu Photonics, Hamamatsu, Japan) and analyzed with Aperio ImageScope (version 11.1, Leica Microsystems, Amsterdam, The Netherlands).
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