Biopsies were fixed in 4% paraformaldehyde and stored at 4°C. At minimum 7 days prior to embedding in Tissue Tek (Sakura Finetek) biopsies were placed in 70% ethanol. 3.5 µm thick sections were deparaffinized in 2*10 min Tissue clear and dehydration. Serial sections of the biopsies were analyzed to ensure a thorough overview of B cell distribution throughout the collected tissue specimen. Sections were stained by Mayers Hematoxylene/Erosin (Histolab) according to the manufacturer’s protocol. Sections stained with CD20 antibody (M07755, clone L26, DAKO) was pre-treated with 98°C TEG-buffer (10 mM Tris, 0.5 mM EGTA, pH 9.0) for 15 min and 1% hydrogen peroxide (Merck) for 15 min. Sections were incubated with CD20 antibody (1:500) overnight at 4°C. Visualization was performed with Envision Mouse (K4001, DAKO) and NovaRED (VECTOR) according to manufacturer’s protocol. Sections were mounted using Tissue Tek tissue mount (Sakura Finetek).
Envision mouse
Manufactured by Agilent Technologies
Sourced in Denmark, United States, Sweden
The Envision Mouse is a high-performance optical mouse designed for laboratory use. It features a precise optical sensor and a comfortable ergonomic design to provide accurate and reliable cursor control for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Novared, by Vector Laboratories (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Tissue tek, by Sakura Finetek (1 mentions)
Decloaking chamber, by Biocare Medical (1 mentions)
Dab chromogen, by Agilent Technologies (1 mentions)
Qimaging camera, by Nikon (1 mentions)
Epitomics, by Abcam (1 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Mac 3, by BD (1 mentions)
En vision rabbit, by Agilent Technologies (1 mentions)
Interleukin 6 (il 6), by Santa Cruz Biotechnology (1 mentions)
P stat3, by Abcam (1 mentions)
Diaminobenzidine substrate, by Agilent Technologies (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
5 protocols using envision mouse
1
Immunohistochemical Analysis of B-Cell Distribution
2
Automated Ki-67 Immunohistochemistry Staining
3
Immunohistochemical Analysis of Aortic Inflammation
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Human (n ¼ 10 AAA samples and n ¼ 10 control atherosclerotic aorta samples) and murine tissue sections were deparaffinized and incubated overnight at room temperature with the primary antibody diluted in PBS, 1% BSA, using the following primary antibodies for the human studies: IL-6 (Santa Cruz Biotechnology, USA), IL-6R or CD126 (Abcam, UK) and pSTAT3 (Abcam, UK). Envision mouse or Envision Rabbit (Dako, Denmark) were used as secondary antibody [15] .
Murine sections were incubated with CD45 (BD Pharmingen, USA), MAC3 (BD Pharmingen,USA), MMP9 (Santa Cruz Biotechnology, USA) and Smooth Muscle Alpha Actin (DAKO, Denmark). Further sections were stained with Sirius Red for collagen and Weigert's elastin stain to visualize elastic lamina. Eight slides per animal were used per staining for analysis and only moderate or strongly reactive cells were counted as positive. The slides were blindly evaluated. A mean value for positive staining cells in the eight sections was calculated for each animal.
Murine sections were incubated with CD45 (BD Pharmingen, USA), MAC3 (BD Pharmingen,USA), MMP9 (Santa Cruz Biotechnology, USA) and Smooth Muscle Alpha Actin (DAKO, Denmark). Further sections were stained with Sirius Red for collagen and Weigert's elastin stain to visualize elastic lamina. Eight slides per animal were used per staining for analysis and only moderate or strongly reactive cells were counted as positive. The slides were blindly evaluated. A mean value for positive staining cells in the eight sections was calculated for each animal.
4
Immunohistochemical Analysis of Tumor Markers
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The most representative tumor tissue block was chosen from each sample, and 5 μm sections were attached to poly-L-lysine-coated slides for immunohistochemical staining. The tissue sections were deparaffinized in xylene, rehydrated in alcohol, and immersed in distilled water. The sections were then boiled for ten minutes in citrate buffer solution (10 mM, pH 6.0) in a microwave oven 3 times for epitope retrieval in staining, and the standard streptavidin-biotin immunoperoxidase method was used for immunostaining with Ki-67 antigen, HER-2, ER, PR and p53 according to [67 (link)]. The sources and dilutions of these antibodies and the epitope retrieval methods are listed in Table 1 . Bound antibodies were detected using Envision TM mouse or rabbit (Dako, USA) secondary antibodies after a 45 min incubation at room temperature. Samples were considered HER-2 positive if they scored 3+ according to the appropriate guidelines [63 (link)].
5
Immunohistochemical Detection of RSV
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For unmasking, sections were treated with heat-induced epitope retrieval (HIER). They were placed in HIER buffer (Target Retrieval Solution, pH = 6, DAKO, Sweden) and subjected to heat treatment in HIER Microwave at 750 W for 7 minutes followed by 350 W for 14 minutes and were allowed to stand for 20 min at room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 min at room temperature. Unspecific antigen staining was blocked with 2% bovine serum albumin (Sigma-Aldrich, Sweden AB) for 20 min. The slides were then incubated at room temperature for 45 min with mouse monoclonal antibody anti RSV (clone 5H5, 2G122, 5A6 and 1C3, NCL-RSV3, Novocastra, Leica Microsystems, Sweden) diluted 1:100 in diluents buffer (1% BSA/TBS pH = 7.6). The detection was conducted with the dextran polymer method (EnVisionTM/mouse, DAKO, Sweden). The color was developed with diaminobenzidine substrate (DAB, DAKO, Sweden). Sections were counterstained with haematoxylin. Antibody-omission stained sections served as negative controls for each section. Appropriate positive and negative control sections were included in each run.
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