Paraffin-embedded tissue sections on coated slides were dewaxed in xylene and rehydrated. Frozen sections of mouse tissues were placed directly on slides and then fixed with 10% buffered formalin. All subsequent washes were done with PBS–0.05% Tween 20 (PBS-T). Sections were first blocked in CarboFree (10% normal goat serum for SGRP probing) for 1 h at 37°C. Esterase treatment control slides were exposed to active esterase HE-Fc forms (20 μg/μl) during serum blocking. Sections were further blocked with avidin/biotin (Vector Laboratories, Burlingame, CA) and quenched of active peroxidase activity by the use of Bloxall (Vector Laboratories). Sections were probed with HE-Fc SGRPs (at 20 μg/ml) in complex with biotinylated goat anti-human Fc γ-specific antibody (10:1 molar ratio) overnight at 4°C. Sections were probed with biotinylated plant lectins SNA (Vector Laboratories) and MAH (MAA-II, Vector Laboratories) at 10 μg/ml and 20 μg/ml, respectively, for 3 h at RT. Sections were then exposed to ABC Vectastain strepavidin-HRP, followed by NovaRed HRP substrate (Vector Laboratories). Sections were counterstained in hematoxylin, dehydrated, and embedded in Cytoseal XYL.
Novared hrp substrate
Manufactured by Vector Laboratories
NovaRed HRP substrate is a chromogenic substrate for the detection of horseradish peroxidase (HRP) in immunoassays and other applications. It produces a red-brown colored product upon enzymatic conversion, enabling the visualization of HRP-labeled targets.
Automatically generated - may contain errors
Lab products found in correlation
Bloxall, by Vector Laboratories (1 mentions)
Xylene substitute, by Merck Group (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Bloxall reagent, by Vector Laboratories (1 mentions)
Axioimager microscope, by Zeiss (1 mentions)
Hematoxylin qs, by Vector Laboratories (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
U6382, by Merck Group (1 mentions)
Mayer s hematoxylin, by Merck Group (1 mentions)
5 protocols using novared hrp substrate
1
Histochemical Analysis of Glycan Binding
2
Histological Analysis of Cellular Infiltration
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To analyze the cellular infiltration, 5-μM-thick sections were cut from paraffin-embedded tissue blocks and placed on charged microscope slides for staining. Sections were deparaffinized, rehydrated, and subjected to routine hematoxylin (Sigma) and eosin (Sigma) staining. Representative images were acquired using a Zeiss AxioImager microscope. To determine the phenotype of the cells at the injection site, 5-μM sections from paraffin-embedded tissues were cut, prepared, and blocked as described above. Endogenous peroxidase/phosphatase activity was blocked using Bloxall reagent (Vector Labs). Anti-F4/80 (6640; Abcam) and anti-CD3 (RM-9107; Thermo Scientific) as primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were used. Peroxidase activity was visualized using NovaRed HRP substrate (Vector Labs). Slides were then counterstained with hematoxylin, dehydrated with ethanol, and cleared with xylene substitute (Sigma). Coverslips were mounted with Organo/Limonene mounting medium (Sigma). Representative images were acquired using a Zeiss AxioImager microscope.
3
Immunohistochemical Tissue Analysis
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Liver tissues were fixed in formaldehyde and subsequently embedded in paraffin for sectioning. Four µM thick sections underwent epitope retrieval and were subsequently stained with primary antibodies for 16 h at 4 ˚C. Slides were then exposed to biotin-conjugated secondary antibodies for 45 min and then streptavidin peroxidase for 10 min. Staining was visualized using the Nova Red HRP Substrate (Vector Laboratories). A complete list of antibodies used for immunohistochemical analysis is presented in Supplementary Table 3 of the Supplementary materials.
4
IHC Staining Protocol for Paraffin Tissues
5
Quantifying UCP1 Expression and Adipocyte Size
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At the end of the 4-week intervention period (experiment 1), mice were euthanized by CO2 inhalation, collected organs were weighed, and interscapular BAT (iBAT), gonadal WAT (gWAT), and subcutaneous WAT (sWAT) samples were fixated in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Cross-sections of 5 μm were deparaffinized and treated with 0.3% H2O2 in 40% CH3OH to quench endogenous peroxidases (30 min). Sections were immersed in 10 mM citrate buffer at pH 6.0 (10 min, 97°C), blocked with 5% normal BSA in PBS (30 min, room temperature), and incubated overnight at 4°C with rabbit polyclonal anti-UCP1 (U6382; Sigma-Aldrich; 1:2000 in 1% BSA). After incubation, sections were incubated with HRP-labeled α-rabbit secondary antibody (K4003; DAKO EnVision™; 30 min, room temperature), which were then visualized by NovaRED™ HRP substrate (Vector Labs; 7 min, room temperature). Sections were counterstained with Mayers Hematoxylin (1.09249; Merck; 1:4 in H2O, 45 s, room temperature). Expression of UCP1 was quantified in iBAT and sWAT using ImageJ software (National Institutes of Health; version 1.52a), and stained area was expressed as a relative percentage of total lean area. Also using this software, the average adipocyte size was determined in gWAT and sWAT. Because of technical problems n = 6 per group remained for histological analysis.
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