Tissue sections (4 μm) were deparaffinized, rehydrated, washed and heated in 0.01 M citrate buffer (pH 6) at 125 °C for 5 min and at 90 °C for 10 min in a decloaking chamber (Biocare Medical). Sections were stained using the rabbit anti-PMCA2 ATPase polyclonal antibody (1:300; PA1-915 Thermo-Fisher Scientific) or β-casein monoclonal antibody (1:100; sc-53189, Santa Cruz), and the MACH-1 Universal HRP-Polymer Detection Kit (Biocare Medical) according to the manufacturer’s instructions. Nuclei were counterstained with hematoxylin using a Varistain Gemini ES Automated Slide Stainer (Thermo Fisher Scientific). The negative and positive controls were no primary antibody and cerebellar tissue, respectively. Stained tissue sections were scanned at 20× magnification using a ScanScope XT Digital Slide Scanner (Aperio), and evaluated by a blinded pathologist (LdS) using the following criteria: (1) positive: intense plasma membrane staining with or without cytoplasmic staining; (2) negative: cytoplasmic or no staining (since PMCA2 is a plasma membrane Ca2+-transporter).
Mach 1 universal hrp polymer detection kit
Manufactured by Biocare Medical
Sourced in United States
The MACH 1 Universal HRP-Polymer Detection Kit is a laboratory reagent used for immunohistochemical staining procedures. It provides a detection system that utilizes a horseradish peroxidase (HRP)-conjugated polymer backbone to amplify the signal from primary antibody binding in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Varistain gemini es automated slide stainer, by Thermo Fisher Scientific (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
Tbs autowash buffer, by Biocare Medical (1 mentions)
Prism v6, by GraphPad (1 mentions)
Aperio at turbo slide scanner, by Leica (1 mentions)
Ab15086, by Abcam (1 mentions)
9 protocols using mach 1 universal hrp polymer detection kit
1
Immunohistochemical Detection of PMCA2 ATPase
2
Immunohistochemical Analysis of Paraffin-Embedded Tissues
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Consecutive sections cut from paraffin-embedded blocks were deparaffinized in xylene, rehydrated through decreasing concentrations of alcohol, and rinsed in Tris-buffered saline (TBS, pH 7.6). Samples were then autostained using the IntelliPATH system (Biocare Medical, Concord, CA, USA). Incubation with monoclonal antibodies (mAb) for 30 min was followed by visualization using the MACH 1 Universal HRP-Polymer Detection Kit (Biocare Medical, Concord, CA, USA). For routine histological analysis, sections were counter-stained with haematoxylin (BioCare Medical, Concord, CA, USA). For TF detection, a newly developed and evaluated mAb was used (HPA049292; kind gift from Human Protein Atlas, Uppsala, Sweden) (13 (link)). For the identification of macrophages, we used a mAb against CD68 (Clone PG-M1; DAKO, Glostrup, Denmark). The samples were finally examined with a Leica DRMB microscope.
3
Immunohistochemical and Immunofluorescence Analysis of Cryopreserved Liver Tissues
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Cryopreserved liver tissues were fixed in cold acetone, washed in PBS, and the endogenous peroxidase activity blocked with 3% hydrogen peroxide or 30 minutes. Then, a Mach 1 Universal HRP Polymer Detection Kit (Biocare Medical, USA) was used according to the manufacture’s recommendations. The slides were incubated with rabbit anti-mouse NOS-2 ntibody (1:400, Santa Cruz) and counterstained with Mayer’s hematoxylin. For immunofluorescence, the slides were incubated in 0.5% saponin in PBS for 15 minutes, and nonspecific sites blocked with 1% BSA for 30 min RT. The slides were incubated with a FITC-conjugated anti-CD11c antibody and Alexa fluor 594 anti-iNOS antibody (1:100, Biolegend) overnight at 4°C. The sections were washed, and the nucleus stained and mounted with Prolong. The images were analyzed using the Leica SP5 (Leica Microsystems).
4
Immunohistochemistry for Wnt Signaling Markers
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Sections (4 μM) were prepared on superfrost microscope slides (Menzel gläser, Germany). The sections were deparaffinized in xylene twice for 5 minutes, rehydrated in a descending series of ethanol (99%, 96%, and 70%), and followed by washes in distilled water. Antigen retrieval was achieved by heating the samples in retrieval buffer with low pH 6.2 (DIVA Decloacer Biocare Medical, USA) or high pH 9.5 (BORG Decloacer Biocare Medical, USA) using Decloaking chamber tm NxGen (Biocare Medical, USA) at 110°C for 15 minutes. Then the sections were washed in wash buffer (TBS Auto wash buffer, Biocare Medical, USA). Staining was performed using MACH1 Universal HRP polymer detection kit and Immunostainer Intellipath (Biocare Medical, USA) according to the manufacturer's protocol. The slides were incubated 30 minutes with three primary antibodies (anti-axin, anti-APC and anti-β-catenin). The antibodies and retrieval buffers used are presented in Table 2 . The slides were counterstained with Mayer's Hematoxyline (Histolab Products, Sweden), and mounted with PERTEX for examination.
5
Immunostaining of Smooth Muscle Cells
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HCASMCs were seeded on a glass‐coverslips, harvested and then fixed in 4% paraformaldehyde. Cell permeabilization was performed with 0.3% Triton X‐100 on ice for 10 min. Then, samples were blocked in PBS, 0.02% NP‐40, 1% bovine serum albumin (BSA) for 30 min. Smooth muscle actin (ACTA2) was visualized using a monoclonal primary antibody (Abcam, Ca# ab32575). Thereafter, MACH 1 Universal HRP‐Polymer Detection kit (BIOCARE Medical, Ca# M1U539) was utilized, following the manufacturer’s indications. After DAB development, haematoxylin counterstaining was performed, and images acquired with the VS120 DotSlide slide scanner (Olympus).
6
Histopathological and Immunohistochemical Analysis of Pandemic Influenza and COVID-19 Lung Autopsies
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Formalin-fixed and paraffin-embedded lung autopsy specimens from patients who died of pandemic influenza A(H1N1) or COVID-19 (N=2 patients per group) were obtained from the Pathology Department of the INER. Sections of 3-5μm were processed for hematoxylin-eosin (H&E) staining for histopathological analysis. For immunohistochemistry (IHQ), lung sections were mounted on silane-covered slides, deparaffinized in xylenes, and hydrated with a series of graded alcohol-to-water dilutions. The endogenous peroxidase was blocked with 3% hydrogen peroxide for 30 minutes. Sections were incubated overnight at room temperature with optimal dilutions (1:100) of the following antibodies: anti-IFN-γ (Anti-Interferon gamma antibody, ab9657, Abcam, UK), anti-IL-1β (IL-1β Antibody (H-153): sc-7884, Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-IL-4 (IL-4 Antibody (OX81): sc-53084, Santa Cruz Biotechnology Inc., Santa Cruz, CA), and anti-IL-17A (Anti-IL-17 antibody (ab91649), Abcam, UK). Secondary biotinylated antibodies labeled with peroxidase were added, and those attached were revealed with diaminobenzidine (DAB) for 5 minutes (MACH 1 Universal HRP-Polymer Detection Kit, Biocare Medical, LLC). Slides were counter-stained with hematoxylin.
7
SASH1 Expression in Breast Cancer
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SASH1 protein expression in breast cancer was investigated by IHC analysis of the Queensland follow-up (QFU) resource, comprising TMAs of 449 invasive breast carcinomas (sampled in duplicate) and associated clinical data, including survival outcomes of over 20 years [33 (link)]. The use of patient data and clinical samples in this study were approved by human research ethics committees of the University of Queensland and the Royal Brisbane and Women's Hospital (RBWH).
Four μm TMA sections were processed in a decloaker for antigen retrieval in EDTA buffer (pH 8.8) for 15 minutes, and then IHC was performed using an anti-SASH1 antibody (Sigma Prestige HPA029947; 1:850), and the Mach 1 Universal HRP-Polymer Detection kit (Biocare Medical). Haematoxylin-counterstained, mounted sections were then scanned at 40 x magnification on an Aperio AT Turbo slide scanner (Leica Biosystems). Digital images of individual tissue cores were scored by a qualified Pathologist (AMM) according to tumour cell nuclear staining intensity. Using the maximum score of duplicate tissue cores for each case, associations between SASH1 expression and clinicopathologic variables were investigated using chi-square and log-rank tests (GraphPad Prism v6).
Four μm TMA sections were processed in a decloaker for antigen retrieval in EDTA buffer (pH 8.8) for 15 minutes, and then IHC was performed using an anti-SASH1 antibody (Sigma Prestige HPA029947; 1:850), and the Mach 1 Universal HRP-Polymer Detection kit (Biocare Medical). Haematoxylin-counterstained, mounted sections were then scanned at 40 x magnification on an Aperio AT Turbo slide scanner (Leica Biosystems). Digital images of individual tissue cores were scored by a qualified Pathologist (AMM) according to tumour cell nuclear staining intensity. Using the maximum score of duplicate tissue cores for each case, associations between SASH1 expression and clinicopathologic variables were investigated using chi-square and log-rank tests (GraphPad Prism v6).
8
Immunohistochemical Evaluation of Tumor Protein Expression
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The TMA slides were pretreated in microwave oven (800 W, pH 9, 10 mmol EDTA). The primary rabbit anti-human antibody was used in a dilution of 1:500 (18865, Immuno-Biological Laboratories, Gunma, Japan). For detection the MACH1 Universal HRP polymer detection kit was used (Biocare Medical, Concord, CA). Both membranous and cytoplasmic staining was assessed by evaluating the percentage of positively stained cells. Three punctures of separate tumor areas were assessed. The percentage of positivity was graded into four groups: 0 ≤ 1%, 1 = 1-10%, 2 = 11-50%, 3 = 51-80%, 4 = 81-100%. A limit for cellular positivity was when 1% or more of tumor cells expressed positivity. The staining was evaluated by two experienced pathologists (AT, YS).
9
Biomarker Expression in Metastatic Tumor Tissue
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The expression of PTEN, PI3K subunit p110a, c-MET, and CAIX was assessed by immunohistochemistry (IHC) on 4 consecutive 2-µm thick tissue sections of formalinfixed, paraffin-embedded (FFPE) specimens. Sections were immunostained with biomarker-specific antibodies as follows: PTEN (Cell Signalling 9559; dilution 1:100), PI3K subunit p110a (Cell Signalling 4249; dilution 1:200), CAIX (Abcam AB15086; dilution 1:2,000), and c-MET (Assay Designs ADI-905-076; dilution 1:100). Slides were then incubated with the secondary antibody using the MACH1 Universal HRP-Polymer Detection kit (Biocare Medical). Staining was performed with 3,3' diaminobenzidine (DAB) as a chromogen and sections were then counterstained with hematoxylin. For each batch, positive and negative control slides without the primary antibody were also included.
Biomarker analysis was performed on archived tissue from metastatic sites and, when available, on tissue from the correspondent primary tumors. For patients with archived tissues from 2 or more metastatic sites, all tumor specimens were analyzed. Nevertheless, only the results from the biomarker analysis conducted on tumor tissue obtained before the start of the bevacizumab-based treatment were used for correlation with the clinical data. All the analyses were performed by pathologists who were blinded to baseline patient characteristics and clinical outcomes.
Biomarker analysis was performed on archived tissue from metastatic sites and, when available, on tissue from the correspondent primary tumors. For patients with archived tissues from 2 or more metastatic sites, all tumor specimens were analyzed. Nevertheless, only the results from the biomarker analysis conducted on tumor tissue obtained before the start of the bevacizumab-based treatment were used for correlation with the clinical data. All the analyses were performed by pathologists who were blinded to baseline patient characteristics and clinical outcomes.
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