The brain sections harvested from all animals were processed for Fos immunohistochemistry and quantified as described previously (e.g., Koya et al., 2009b (link); Bossert et al., 2011 (link); Pfarr et al., 2015 (link)). Fos antibody (1:2000 dilution) from Cell Signaling Technology (Cat# 2250S, Danvers, MA, USA; RRID: AB_2247211 ) was used. The sections were developed using an ImmPRESS HRP (Peroxidase) Polymer Kit from Vector Laboratories (Cat# MP-7451, Burlingame, CA, USA; RRID:AB_2631198 ) and diaminobenzide. β-galactosidase antibody (1:1000 dilution) from Santa Cruz Biotechnology (Cat# sc-65670, Dallas, TX, USA; RRID:AB_831022 ) was used. Additional brain sections were processed for X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) histochemistry to validate the presence of β-galactosidase as described previously (e.g., Koya et al., 2009b (link); Bossert et al., 2011 (link); Pfarr et al., 2015 (link)). We purchased X-gal kit (Cat# XGAL-0100;) from Rockland Immunochemicals Inc. (Pottstown, PA).
Immpress hrp peroxidase polymer kit
Manufactured by Vector Laboratories
Sourced in United States
The ImmPRESS HRP (Peroxidase) Polymer Kit is a ready-to-use detection system that uses a polymer conjugated with horseradish peroxidase (HRP) enzyme. The kit is designed for immunohistochemical and immunocytochemical staining applications.
Automatically generated - may contain errors
6 protocols using immpress hrp peroxidase polymer kit
1
Fos Immunohistochemistry and β-Galactosidase Histochemistry
2
Fibrin Deposition Visualization in Perfused Tissues
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Mice were perfused via both right and left ventricles with PBS. Tissues were collected and embedded in paraffin, then sectioned at 5 mm. Anti-fibrin antibody 59D8 (kindly provided by Dr. Hartmut Weiler at Medical College of Wisconsin and Dr. Rodney M. Camire at the University of Pennsylvania) at 4 mg/ml was used for staining fibrin deposition, with M.O.M.® (Mouse on Mouse) ImmPRESS® HRP (Peroxidase) Polymer Kit (Vector, Cat#MP-2400) from vector laboratories according to manufacturer’s instruction for developing positive staining.
3
Tyramide-based Signal Amplification for L1 Detection
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L1 signals were detected using a tyramide-based signal amplification (TSA) method (73 (link)). A detailed protocol is available at https://www.protocols.io/view/untitled-protocol-i8cchsw .
For immunohistochemistry, tissue sections were deparaffinized in xylenes and rehydrated in 100%, 95%, 70%, and 50% ethanol and then in water. Antigen unmasking was performed by heating with 10 mM citrate buffer (pH 6) for 20 min. Blocking was performed with 2.5% horse serum in PBST for 1 h at room temperature (RT). Slides were incubated in primary antibody (BrdU, MCM7; Thermo Scientific, Fremont, CA) at 4°C overnight in a humidified chamber. M.O.M. ImmPRESS HRP (peroxidase) polymer kit (MP-2400; Vector) was applied the next day for 1 h at room temperature for secondary antibody incubation. Slides were then incubated with 3,3′-diaminobenzidine (Vector Laboratories), and counterstained with hematoxylin. All images were taken with a Zeiss AxioImager M2 microscope using AxioVision software, version 4.8.2.
For immunohistochemistry, tissue sections were deparaffinized in xylenes and rehydrated in 100%, 95%, 70%, and 50% ethanol and then in water. Antigen unmasking was performed by heating with 10 mM citrate buffer (pH 6) for 20 min. Blocking was performed with 2.5% horse serum in PBST for 1 h at room temperature (RT). Slides were incubated in primary antibody (BrdU, MCM7; Thermo Scientific, Fremont, CA) at 4°C overnight in a humidified chamber. M.O.M. ImmPRESS HRP (peroxidase) polymer kit (MP-2400; Vector) was applied the next day for 1 h at room temperature for secondary antibody incubation. Slides were then incubated with 3,3′-diaminobenzidine (Vector Laboratories), and counterstained with hematoxylin. All images were taken with a Zeiss AxioImager M2 microscope using AxioVision software, version 4.8.2.
4
Multiparametric Analysis of Liver Inflammation
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Frozen mouse liver samples (5 μm) were stained with rat anti-CD68 Abs, rat anti-Ly6G Abs, rabbit anti-H3Cit Abs, sheep anti-CC1 Abs, or rabbit anti-S1PR2 Abs. Hepatic Ly6G+ cells were scored semiquantitatively by blinded counting of cells in 10 HPF/sections (×400). Isolated neutrophils were stained using sheep anti-CC1 Abs, rat anti-Ly6G Abs, goat anti-MPO Abs, or rabbit anti-H3Cit Abs. Signals were visualized with secondary Alexa Fluor Abs. For S1P detection, formalin-fixed, paraffin-embedded liver tissues (5 μm thickness) were stained using mouse anti-S1P Abs with the M.O.M. (Mouse on Mouse) ImmPRESS HRP (peroxidase) Polymer Kit (MP-2400, Vector Laboratories).
5
Immunohistochemical Analysis of Lung Tissue
6
Immunohistochemical Analysis of Fibrin Deposition
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Lungs are flushed through the right ventricle of the heart with warm PBS to be clear of blood, fully inflated with 10% formalin via intratracheal instillation, and excised en bloc after trachea ligation. Lungs are subsequently fixed in 10% formalin overnight and included in paraffin. Lung sections are cut at 5 µm and mounted on glass slides. Sections are incubated in an oven at 80°C for 30 min, deparaffinized with xylene and cleaned in ethanol and water. Deparaffinized sections are placed overnight in antigen retrieval solution (10 mM sodium citrate buffer, pH= 6) at 65–80°C. Sections are then removed from the oven to room temperature and allowed to cool for about 20 min, before proceeding with immunostaining. We use a primary anti-fibrin mouse monoclonal antibody (clone 59D8, Cat. No. MABS2155, Sigma-Aldrich) and a commercial kit (M.O.M.® ImmPRESS® HRP (Peroxidase) Polymer Kit, MP-2400, Vector), that contains a mouse Ig blocking reagent paired with a specialized, ready-to-use, one-step M.O.M. ImmPRESS Peroxidase Polymer reagent. Detection of fibrin deposition is achieved using diaminobenzidine (DAB) substrate (ImmPACT® DAB Substrate Kit, Peroxidase (HRP), SK-4105, Vector), which produces a brown reaction product. Sections are counterstained with hematoxylin, dehydrated, cleared in xylene and mounted (Vecta Mount, H-5000, Vector) for microscopic analysis.
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