Following the last imaging sessions on day 12, mice were euthanized by isoflurane overdose and then perfusion-fixed with 4% paraformaldehyde. The right limb muscle of mice was excised and paraffin-embedded. Embedded tissues were sectioned (5 μm in thickness) every 400 μm to ensure entire sampling of the tissue. These sections were stained with hematoxylin and eosin for general tissue morphology, PPB and nuclear fast red counterstain to identify the presence of ferumoxytol, or F4/80 immunohistochemical staining to identify macrophages. For F4/80 staining, sections underwent antigen retrieval in sodium citrate buffer, permeabilized using 0.4% Triton X-100 (Sigma-Aldrich, Oakville, Ontario, Canada), followed by overnight incubation in rat antimouse F4/80 primary antibody [1:100 dilution] (ab16911, Abcam). The next day, sections were incubated with biotinylated goat antirat IgG antibody [1:300 dilution] (BA-9401, Vector Laboratories) and then processed with ABC solution (PK4000, Vector Laboratories, Burlington, Ontario, Canada). Lastly, the slides were incubated in 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate solution (SK-4100, Vector Laboratories) and counterstained with hematoxylin. Histological images were acquired on the EVOS Imaging System (M7000, Thermo Fischer Scientific).
Ba 9401
Manufactured by Vector Laboratories
Sourced in Canada, United States, United Kingdom
The BA-9401 is a laboratory instrument designed for the detection and quantification of biological analytes. It utilizes advanced spectroscopic techniques to provide accurate and reliable measurements. The core function of the BA-9401 is to support research and analysis activities in life science and biotechnology applications.
Automatically generated - may contain errors
Lab products found in correlation
Image pro plus 7, by Media Cybernetics (2 mentions)
Mab386, by Merck Group (2 mentions)
Mca1957ga, by Bio-Rad (2 mentions)
Ab6640, by Abcam (2 mentions)
Ba 1000, by Vector Laboratories (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Ab16911, by Abcam (1 mentions)
Evos imaging system, by Thermo Fisher Scientific (1 mentions)
Pk 4000, by Vector Laboratories (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Sk 4100, by Vector Laboratories (1 mentions)
Superfrost plus micro slide, by Avantor (1 mentions)
Cytoseal 60, by Thermo Fisher Scientific (1 mentions)
Bx41 bright field microscope, by Olympus (1 mentions)
Mca74g, by Bio-Rad (1 mentions)
Ab64693, by Abcam (1 mentions)
Vecstatin elite abc kit, by Vector Laboratories (1 mentions)
Ab15323, by Abcam (1 mentions)
Ab214421, by Abcam (1 mentions)
Ab16669, by Thermo Fisher Scientific (1 mentions)
11 protocols using ba 9401
1
Histological Analysis of Muscle Tissue
2
Quantifying Myelination Developmental Trajectory
3
Immunohistochemical Detection of CD11b
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Lung and colon tissues embedded in paraffin were sectioned and rehydrated. The tissues were antigen retrieved in tri-sodium citrate buffer containing 0.2% tween 20 (pH 6.0) with an autoclave for 1 min. 3% H2O2 solution was treated for 10 min to block endogenous peroxidase activity. The tissues were washed in PBS and blocked with 1.5% bovine serum albumin (BSA) containing 0.2% triton X-100 for 1h at room temperature. Rat anti-mouse CD11b primary antibodies (MCA74G) (Abd Serotec; 1:200) and biotinylated anti-Rat antibodies (BA-9401) (Vector laboratories; 1:1,000) were diluted in 0.5% BSA. The slides were overnight incubated with primary antibodies at 4°C and incubated with secondary antibodies for 1h at room temperature. The avidin-biotin complex kit (Vectastain ABC kit; PK-6102) (Vector Laboratories, Burlingame, CA, USA) and diamminobenzidine-HCl (DAB) kit (SK-4100) (Vector Laboratories, Burlingame, CA, USA) were used to visualize the tissue. The slides were mounted and observed under the microscope.
4
Quantifying Myelination in Brain Sections
5
Immunohistochemical Analysis of Tissue Markers
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Immunohistochemistry (IHC) was performed using the Vecstatin Elite ABC kit (Vector Labs) and following the manufacturer’s instructions. Briefly, sections were fixed in acetone, endogenous peroxidase quenched in 0.3% hydrogen peroxide in methanol, and processed for immunostaining for CD68 (#MCA1957GA, Bio-Rad), inducible nitric oxide synthase (“iNOS”, antibody #ab15323, Abcam), mannose receptor (“CD206”, antibody #ab64693, Abcam) or α-smooth muscle actin (“αSMA”, antibody #ab5294, Abcam). After treatment with secondary antibodies (for CD68: biotinylated goat anti-rat antibody #BA-9401, Vector laboratories; for iNOS, CD206 and αSMA: biotinylated goat anti-rabbit antibody, Vecstatin Elite ABC kit, Vector laboratories), sections were incubated with Vectastain ABC reagent and peroxidase substrate solution (# SK-4100, Vector laboratories). Counterstaining was with hematoxylin. Negative controls were performed by substituting the primary antibody with non-immune IgG from the same species and at the same concentration. Under these conditions, nonspecific immunostaining was not detected. Stained areas were captured (Micropublisher 3.3 Megapixel Cooled CCD Color Digital Camera) and measured (NIS Elements D).
6
Quantifying Immune Cell Populations in Intestinal Tumors
7
Immunohistochemical Analysis of Cellular Markers
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Antigen retrieval was performed with Tris-EDTA [platelet-derived growth factor receptor (PDGFR)β only], endogenous peroxidases were quenched with 3% H2O2, Avidin/Biotin block was applied (SP-2001; Vector Laboratories) before blocking with 20% goat serum (GR1/PDGFRβ) or rabbit serum (F4/80). Primary antibodies were applied for 2 hours at room temperature (PDGFRβ, dilution 1:500; ab32570; Abcam, Cambridge, UK) or overnight at 4°C (GR1, dilution 1:750; MAB1037; R&D, Abingdon, UK; F4/80, dilution 1:200; ab6640; Abcam). Secondary antibody (PDGFRβ–biotinylated goat anti-rabbit, dilution 1:1000; BA-1000; Vector Laboratories; GR1–biotinylated goat anti-rat, dilution 1:1000; BA-9401; F4/80–biotinylated rabbit anti-rat, dilution 1:200; BA-4001) was applied for 30 minutes at room temperature. Detection was performed by using the Elite Vectastain ABC kit and 3,3′-diaminobenzidine, before counterstaining, dehydration, and mounting. For each sample, 10 sequential fields were acquired at ×20 magnification, and the percentage of positive staining was calculated by using FIJI.36 (link)
8
Histological Assessment of Cardiac Pathology
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As previously described [21 (link)], hearts were perfused through the left ventricle with 1× phosphate buffered saline (PBS, Thermo Scientific, Waltham, MA, USA), followed by 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), cut and embedded in optimal cutting temperature compound (OCT, Tissue-Tek, 4583, Torrance, CA, USA). This was followed by frozen-sectioning on a microtome-cryostat (10 µm) starting from where the aorta exits the ventricle and moving towards the aortic sinus. Sections were stained with ORO or trichrome (Gomori’s Trichrome Stain Kit, 87020, Thermo Scientific, Walthman, MA, USA), or subjected to immunohistochemical (IHC) analysis with CD68 antibody (1:300; MCA1957GA, Bio-Rad, Hercules, CA, USA) for 3 h at room temperature prior to incubating with a biotinylated goat anti-rat antibody (BA-9401, Vector laboratories, Burlingame, CA, USA) for 1 h. Images were taken at 4X using an Olympus SZX7-TR30 microscope (Tokyo, Japan) and quantified CellSens Standard program.
9
Immunohistochemical Analysis of Apoptosis and Macrophages in Intestinal Tissue
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Sections (5 µm) of paraffin-embedded small intestinal tissue were sectioned and used for immunohistochemistry. Following de-parafinization and rehydration, tissue sections were treated with 1% hydrogen peroxide in methanol to block endogenous peroxidases. Subsequently, slides were treated using heat-induced antigen retrieval in 0.01 M citrate acid buffer (pH 6) followed by incubation with a rabbit polyclonal anti-active caspase-3 (CC3) antibody (AF835: R&D Systems). Visualization of caspase-3 positivity was via a peroxidase-labelled anti-rabbit EnVision secondary antibody (Dako) and 3,3′-diaminobenzidine followed by counterstaining with haematoxylin. For macrophage staining, an antibody against F4/80 antigen (ab6640: Abcam) was employed using biotinylated anti-rat (BA-9401) and avidin–biotin reagent (PK-6100; Vector Laboratories).
10
Quantifying Brain Vessel Density
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Brain tissue sections were stained for blood vessel marker CD31 (AF3628, R&D Systems), VCAM‐1 (1510‐14, Southern Biotech), and the macrophage/microglia marker Iba‐1 (ab5076, Abcam, Cambridge, UK), as described previously.
45 (link),
46 (link),
47 (link) Secondary antibodies used were biotinylated horse anti‐goat IgG (BA‐9500, Vector Laboratories, Peterborough, UK) and biotinylated goat anti‐rat IgG (BA‐9401, Vector Laboratories). Sections were also stained for the angiogenic marker integrin αvβ3 (ab78289, Abcam) using Mouse on Mouse Basic Kit (BMK‐2202, Vector Laboratories) in accordance with the protocol described by the manufacturer.
Microvessel density (CD31 positive area fraction) was quantified for the area encompassing the metastatic foci in the injected striatum, and also for the contralateral striatum, as the percentage of area covered using the ‘Positive Pixel Count 2004‐08‐11’ algorithm in ImageScope (Leica Biosystems). The parameters used were moderately stained pixel intensity, between 202 and 185, and strongly stained pixel intensity, less than 10.
45 (link),
46 (link),
47 (link) Secondary antibodies used were biotinylated horse anti‐goat IgG (BA‐9500, Vector Laboratories, Peterborough, UK) and biotinylated goat anti‐rat IgG (BA‐9401, Vector Laboratories). Sections were also stained for the angiogenic marker integrin αvβ3 (ab78289, Abcam) using Mouse on Mouse Basic Kit (BMK‐2202, Vector Laboratories) in accordance with the protocol described by the manufacturer.
Microvessel density (CD31 positive area fraction) was quantified for the area encompassing the metastatic foci in the injected striatum, and also for the contralateral striatum, as the percentage of area covered using the ‘Positive Pixel Count 2004‐08‐11’ algorithm in ImageScope (Leica Biosystems). The parameters used were moderately stained pixel intensity, between 202 and 185, and strongly stained pixel intensity, less than 10.
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