To detect citrullinated histone and MPO in the spleen, specimens were fixed in 10% formalin for 24 h. Paraffin-embedded spleen sections were incubated at 70 °C for 1 h and deparaffinized in Xylene for 10 min twice. Samples were incubated in boiling citric acid (0.01 M, pH. 6.0) for 30 min, then cooled to room temperature for antigen retrieval. Samples were incubated with anti-citrullinated histone antibody (1:100) or anti-MPO antibody (1:100) at 4 °C overnight, then incubated with HRP-conjugated secondary antibody (1:100) for 1 h at room temperature; HRP activity was determined using the Pierce® Peroxidase Detection Kit (Thermo Fisher Scientific, #36000). Samples were counterstained with hematoxylin, and DAB signal was detected and quantified using Aperio ImageScope V9 and MetaMorph softwares.
Pierce peroxidase detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Peroxidase Detection Kit is a reagent set designed to detect the presence of peroxidase enzyme activity in biological samples. The kit provides the necessary components, including a substrate solution, to facilitate the colorimetric or chemiluminescent detection of peroxidase-labeled targets.
Automatically generated - may contain errors
Lab products found in correlation
Dm6b widefield microscope, by Leica (2 mentions)
Dfc450 c digital camera, by Leica (2 mentions)
Dxm1200, by Nikon (1 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions)
Anti ap 2α, by Santa Cruz Biotechnology (1 mentions)
Eclipse e1000, by Nikon (1 mentions)
Horseradish peroxidase goat anti rabbit secondary antibody, by Abcam (1 mentions)
6 protocols using pierce peroxidase detection kit
1
Spleen Citrullinated Histone and MPO Detection
2
Immunohistochemistry of Metastatic Tumor Model
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Immunohistochemistry (IHC) was performed using the Ptenlox5/lox5;SSM2/SSM2 metastatic mouse model. Tissue was stained using a rabbit anti-human/mouse polyclonal antibody targeting either Oncostatin M Receptor (OSMR) (ABclonal, Woburn, MA, USA. Cat.# A6681) or eGFP (Thermo Fisher, Carlsbad, CA, USA. Cat.# CAB4211). All other reagents were obtained from the Pierce Peroxidase Detection Kit (Thermofisher, Carlsbad, CA, USA. Cat.# 36000) and protocol was followed according to the manufacturer’s protocol. Briefly, a goat anti-rabbit secondary antibody was applied to the tissues, followed by an anti-goat strep-HRP tertiary antibody. Samples were counterstained with hematoxylin, dehydrated, and mounted. Slides were analyzed through a Leica DM6B widefield microscope and imaged with the attached Leica DFC450-C digital camera.
3
Histopathological Analysis of Flavivirus Infection
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Following euthanasia, tissues were collected and stored in 10% buffered formalin prior to standard processing and paraffin embedding. Sections were cut at 5 μm and stained by routine hematoxylin-eosin (H&E) for histopathological analysis or by immunohistochemistry (IHC) as follows. Following deparaffinization, heat induced antigen retrieval was accomplished by immersing slides in 10 mM sodium citrate buffer (pH 6.0) for 15 minutes. Slides were stained using the Pierce Peroxidase Detection Kit (Thermo Scientific). The primary antibody was the pan-flavivirus clone D1-4G2-4-15. PBS-inoculated mice served as negative controls for IHC and H&E. For immunostaining, negative control slides were prepared using tissues from PBS-inoculated mice as described above, as well as tissues from infected mice without primary antibody. Slides were analyzed by a board-certified pathologist.
4
Immunohistochemical Staining Procedure for AP2α
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For immunohistochemical staining, the slides were dewaxed and rehydrated. After heat-mediated antigenic retrieval using a pH 6.0 sodium citrate buffer for 30 min in thermostatic water bath, the sections were treated according to the manufacturer's instructions of the Pierce Peroxidase Detection kit (Thermo Scientific, Rockford, IL, USA). The polyclonal rabbit anti -AP2α (Santa Cruz) was used as primary antibody at a dilution of 1:200 and the goat anti-rabbit IgG-HRP (656120; Life Technologies) at 1:3000 dilution as secondary one. Staining was visualized using the DAB chromogen (Pierce) and the sections were counterstained with Harris Modified Hematoxylin. Negative controls were performed by omission of the primary antibody in each experiment. Slides were evaluated using a Nikon Eclipse E1000 equipped with a Nikon DXM 1200 digital camera with dedicated acquisition software (Nikon ACT-1 v. 2.1; all from Nikon Instruments, Campi Bisenzio, Firenze, Italy).
5
Measuring Bone Marrow Adipocyte Density
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To examine the effect of dietary hempseed on the density of bone marrow adipocytes, immunohistochemistry staining for the marker PRDM16 protein was employed. This zinc finger transcription factor plays an important role in the differentiation of adipocytes within the bone marrow [45 (link),46 (link),47 (link)]. Tissue was stained using a rabbit anti-human polyclonal antibody targeting PRDM16 (ThermoFisher Scientific, Waltham, MA, USA; catalog #PA5-20872). All other reagents were obtained from the Pierce Peroxidase Detection Kit (ThermoFisher Scientific; catalog #36000) and the protocol was followed according to the manufacturer’s instructions. Briefly, a goat anti-rabbit secondary antibody was applied to the tissues, followed by an anti-goat strep-HRP tertiary antibody. Samples were counterstained with hematoxylin, dehydrated and mounted. Slides were analyzed for PRDM16-positive cells (staining intensity) using a Leica DM6B widefield microscope and imaged with the attached Leica DFC450-C digital camera. Quantitative values were obtained by counting PRDM16-positive cells (brown stain) and total cells (blue stain) in the bone marrow and calculating a percentage.
6
Immunohistochemical Evaluation of CREB3L1 in Breast Tumors
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Immunohistochemical staining of CREB3L1 protein was performed on formalin-fixed paraffin-embedded human breast tumor samples obtained from the Manitoba Tumor Bank, according to the manufacturer’s instructions using Pierce Peroxidase Detection Kit (Thermo Scientific, Burlington, ON, Canada). Briefly, sections were de-paraffinized and heated in citrate buffer (pH = 6), followed by an overnight incubation at 4 °C with a rabbit polyclonal anti-CREB3L1 antibody (1:100; Protein Tech, Rosemont, IL, USA 11235-2-AP). Subsequently, slides were incubated with a horseradish peroxidase goat anti-rabbit secondary antibody (1:1000; Abcam, Toronto, ON, Canada ab6721) for 2 hours at room temperature and reacted with 3,3′-Diaminobenzidine (DAB) for 15 minutes. Sections were counterstained with hematoxylin. CREB3L1 staining was evaluated by a pathologist using two criteria, staining intensity (absent = 0, weak = 1, moderate = 2, strong = 3, very strong = 4) and % cells staining positive (0–5 % = 0, 6–49 % = 1, 50–69 % = 2, 70–89 % = 3, 90–100 % = 4). Scores were added together and described as little or no CREB3L1 staining (combined score 0–1), low (scores 2–3), medium (scores 4–5) and high (scores 6–8) CREB3L1 expression, analogous to the Allred system [39 (link)]. In addition, the subcellular location of CREB3L1 was evaluated as nuclear or cytoplasmic.
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