The implanted SF graft was hollowed out together with the skin with surgical scissors and divided in half. In addition, the central part of the graft was cut 4 mm transversely, and the sutured part of the remaining native blood vessel and artificial vascular graft was cut longitudinally. These samples were fixed with ethanol for histological analyses. The fixed samples were embedded in paraffin and processed for hematoxylin and eosin (H&E) and Masson’s trichrome (MTC) staining. Sections for immunohistochemical staining were incubated with primary antibodies, including α-smooth muscle actin (α-SMA; clone 1A4; Sigma-Aldrich, Inc., Tokyo, Japan) and CD31 anti-rat antibody (BD Biosciences, Inc., Tokyo, Japan). The A-SMA sample was incubated with biotinylated anti-mouse immunoglobulin secondary antibody (Vector Laboratories, Inc., Burlingame, CA, USA), and subsequent color development was obtained using Vectastain ABC-AP (Vector Laboratories, Inc., Burlingame, CA, USA). The CD31 sample was incubated with N-Histofine Simple Stain Rat MAX PO (Nichirei Biosciences, Inc., Tokyo, Japan), and subsequent color development was obtained using Vectastain ABC-AP (Vector Laboratories, Inc., Burlingame, CA, USA).
Vectastain abc ap
Manufactured by Vector Laboratories
Sourced in United States
Vectastain ABC-AP is an avidin-biotin complex kit designed for use in immunohistochemical and immunocytochemical staining procedures. It provides a sensitive and reliable method for detecting target antigens in a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Axioskop microscope, by Zeiss (2 mentions)
Cal ex 2, by Thermo Fisher Scientific (2 mentions)
Axiocam camera, by Zeiss (2 mentions)
Streptavidin qdot 655, by Thermo Fisher Scientific (2 mentions)
Nanozoomer sq slide scanner, by Hamamatsu Photonics (2 mentions)
Mouse monoclonal antibody to α smooth muscle cell actin, by Merck Group (2 mentions)
Biotinylated griffonia simplifolia lectin 1, by Vector Laboratories (2 mentions)
α smooth muscle actin α sma clone 1a4, by Merck Group (1 mentions)
N histofine simple stain rat max po, by Nichirei Biosciences (1 mentions)
Dp70 microscope, by Olympus (1 mentions)
Sars cov 2 nucleocapsid rabbit polyclonal antibody, by Thermo Fisher Scientific (1 mentions)
Vector red alkaline phosphatase substrate, by Vector Laboratories (1 mentions)
Haematoxylin qs, by Vector Laboratories (1 mentions)
Anti digoxigenin ap antibody, by Roche (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Cleaved dcp 1, by Cell Signaling Technology (1 mentions)
Nbt bcip, by Vector Laboratories (1 mentions)
Dig rna labelling kit, by Roche (1 mentions)
Nbt bcip, by Roche (1 mentions)
Biotinylated elderberry bark lectin, by Vector Laboratories (1 mentions)
9 protocols using vectastain abc ap
1
Histological Analysis of Vascular Graft Implantation
2
Histological Analysis of Ischemic Limbs
3
SARS-CoV-2 Nucleocapsid Protein Detection
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Tissue sections were stained with SARS-CoV-2 nucleocapsid rabbit polyclonal antibody (Thermo Fisher Scientific). The sections were treated with antigen retrieval solution in a microwave oven and blocked with normal rabbit serum in PBS (pH 7.4). They were then incubated with the rabbit antibody against SARS-CoV-2 NP (1:100 dilution), and subsequently treated with biotin-labelled goat anti-rabbit immunoglobulin (Vector Laboratories, Burlingame, CA, USA), and Vectastain ABC-AP (Vector Laboratories) and Vector Red alkaline phosphatase substrate (Vector Laboratories). The labelled lung sections were counterstained with haematoxylin QS (Vector Laboratories) and observed under an Olympus DP70 microscope (Olympus Corporation).
4
Whole Mount In Situ Hybridization and Antibody Staining
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For whole mount in situ hybridisation gene-specific primers were used (Metabion) to amplify gene fragments via PCR using cDNA synthesised from total RNA. The amplified fragments were subcloned into the pCR4 vector (TOPO-TA Cloning Kit, Invitrogen). In vitro transcription for synthesis of DIG-labelled RNA probes was performed using the DIG RNA Labelling Kit (Roche Applied Science). Whole mount in situ hybridisation was performed as previously described [46 ]. Staining was achieved through application of an Anti-Digoxigenin-AP antibody in combination with NBT/BCIP (Roche Applied Science).
Antibody staining was carried out as previously described [51 ] using antibodies raised against short peptides corresponding to Flipflop1 (NH2-CPKTTKPKAK-CONH2), Flipflop2 (NH2-CSKNTEHKTK-CONH2) (Pineda Antibody-Service) and Cleaved Dcp-1 (#9578, Cell Signaling Technology), respectively. A Biotin-SP-conjugated AffiniPure Anti-Rabbit lgG antibody was used as secondary antibody (Jackson ImmunoResearch Europe Ltd). Staining was carried out using Vectastain ABC-AP (Vector Laboratories) and NBT/BCIP.
Antibody staining was carried out as previously described [51 ] using antibodies raised against short peptides corresponding to Flipflop1 (NH2-CPKTTKPKAK-CONH2), Flipflop2 (NH2-CSKNTEHKTK-CONH2) (Pineda Antibody-Service) and Cleaved Dcp-1 (#9578, Cell Signaling Technology), respectively. A Biotin-SP-conjugated AffiniPure Anti-Rabbit lgG antibody was used as secondary antibody (Jackson ImmunoResearch Europe Ltd). Staining was carried out using Vectastain ABC-AP (Vector Laboratories) and NBT/BCIP.
5
Elderberry Lectin Histochemistry
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Cells fixed in 4 % PFA were washed in 1× PBS and incubated for 10′ at RT in a dual enzyme block solution (Dako, Glostrop, Denmark) followed by wash in 1× PBS, and then incubated with protein block solution for 10′ at RT. The cells were then incubated with biotinylated elderberry bark lectin (1:800) (Vector Labs, Burlingame, CA) for 30 min at RT. Following a wash in 1× PBS, the cells were incubated with vectastain ABC-AP (Vector Labs) complex for 30 min at RT. The ABC-AP complex was prepared fresh according to the instructions provided with the vectastain ABC-AP kit. The cells were washed in 1× PBS to remove the ABC-AP complex and incubated with fast red chromagen staining solution (Dako) for 10′ at RT. The cells were rinsed in water and incubated with gill free hematoxylin for 5′. The slides were rinsed in H2O followed by a gentle wash with 0.5 % ammonium hydroxide. The slides were air dried and mounted on cover slips and the images were captured in tiff format using fluorescence microscope (Nikon) and analyzed.
6
Histological Evaluation of Artificial Vascular Graft Remodeling
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After the removal of the graft, it was immersed in 99.5% methanol for one night. A 4-cm-long section of the artificial vascular graft, which was 4-mm wide and cut in the short-axis direction, was immersed in 99.5% methanol for 30 minutes, dehydrated, immersed in xylene for 90 minutes, and then embedded in paraffin. The embedded samples were sliced in 5-μm sections. The prepared paraffin sections were subjected to hematoxylin and eosin, Masson's trichrome, and Elastica van Gieson staining according to the standard procedures.20 (link),22 (link) For immunohistochemical staining, α-smooth muscle actin mouse anti-monoclonal antibody (Sigma-Aldrich Inc, St Louis, Mo) and CD31 anti-rabbit polyclonal antibody (Abcam Inc, Cambridge, Mass) were used as primary antibodies, followed by anti-mouse IgG biotin (Nichirei Biosciences Inc, Tokyo, Japan) secondary antibodies. Vectastain ABC-AP (Vector laboratories Inc, Burlingame, Calif) was used for color development. By using these staining methods, the ability to remodel autologous tissue postimplantation, ie, replacement with vascular endothelial cells, smooth muscle cells, elastic fibers and collagen fibers, intima thickening with narrowing of the lumen, and inflammatory reaction, was investigated.
7
Histological Analysis of Ischemic Limbs
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Histological analysis was performed on both the ischemic and non-ischemic limbs at day 21 post hind limb ischemia surgery. Mice were euthanized and the tissue was perfused with saline followed by 10% buffered formalin for fixation. The bone was then demineralized in a formic acid based solution (Cal-Ex II, Fisher Scientific) for 48 hours before processing and paraffin embedding. Sections (5 μm thick) were immunostained for vascular smooth muscle cells or endothelial cells. Enzyme treatment was performed in proteinase K (Biolabs 2ug/ml) prior to incubation with primary antibodies. For smooth muscle cells, the sections were stained using a mouse monoclonal antibody to α-smooth muscle cell actin (Sigma) as the primary antibody detected using the avidin-biotin-alkaline phosphatase method (Vectastain ABC-AP, Vector Laboratories) with a hematoxylin counter-stain. Images of the entire section were acquired used the Hamamatsu NanoZoomer SQ slide scanner. To visualize endothelial cells slides were stained with biotinylated Lectin antibody (Biotinylated Griffonia simplifolia Lectin 1, Vector lab), followed by incubation with Streptavidin Qdot 655 (Invitrogen). Images were acquired using the 20X Plan-Neo air objective on a Zeiss Axioskop microscope equipped with an AxioCam camera. ImageJ software (NIH) was used to count the number of vessels for analysis.
8
Lectin-based Glycoprotein Analysis in Mouse Serum
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The protein in 3 μl of mouse serum was precipitated with ice-cold 75% acetone, and the pellet was solubilized in 100 μl of Laemmli sample buffer. Five microliters of solubilized sample (equivalent to 0.15 μl of serum) was separated on a 4 to 15% TGX gradient gel (Bio-Rad), transferred to a polyvinylidene difluoride (PVDF) membrane, and then probed with biotinylated lectins (Sambucus n igra [SNA], Phaseolus v ulgaris erythroagglutinin [E-PHA], Phaseolus vulgaris agglutinin [L-PHA], and Aleuria aurantia [AAL] lectins [Vector Laboratories, Burlingame, CA]) after blocking with 3% BSA in TBS. Probed membranes were incubated with Vectastain ABC-AP (Vector Laboratories), and bound lectin was detected with BCIP/NBT (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD). Alternatively, PVDF membranes were incubated with anti-inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) antibody (ab180139; Abcam, Cambridge, MA), anti-mouse IgM conjugated to horseradish peroxidase (HRP) (A8786; Sigma), anti-ITIH4 antibody (sc-515353; Santa Cruz), goat anti-rabbit IgG conjugated to AP (Promega, Madison, WI), and rabbit anti-mouse IgG conjugated to AP (Promega, Madison, WI). Incubation with secondary antibodies was followed by detection with SigmaFast DAB with metal enhancer (Sigma, Saint Louis, MO) or incubation with the BCIP/NBT phosphatase substrate as appropriate.
9
Somatostatin Receptor Immunostaining
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All pancreases were immunostained for the five SSTRs as previously described (29 (link)). The production and specificity of subtype-specific somatostatin receptor antibodies have been described and discussed earlier (30,31 (link)). Briefly, the immune reaction was amplified by an avidin-biotin complex coupled to alkaline phosphates (Vectastain ABC-AP; Vector Laboratories Inc., Burlingame, CA, USA) and visualized with Vector Red (Vector Laboratories) as substrate (30 (link)). When SSTR-specific antibodies were preincubated with the peptides used for immunization, the immunoreactivity for each receptor was blocked (data not shown) (30 (link)).
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