Coverslips were aminosilanized as described previously [27 ]. Briefly, circular coverslips (No. 1, 25 mm, Fisher Scientific) were cleaned by successive sonication (10 min) in (a) 1% NaPO4, 1% SDS, 1% NaHCO3; (b) ddH2O (double distilled water); (c) acetone; and (d) 1 m NaOH. Cleaned coverslips were rinsed with ddH2O, dehydrated by immersion in methanol, and aminosilanized by incubation (2 periods of 10 min, interrupted by 1 min of sonication) with 1% (v/v) Vectabond (Vector Labs, Burlingame, CA, USA) and 5% (v/v) glacial acetic acid in methanol. The coverslips were extensively rinsed with methanol and then with ddH2O before drying in a dust‐free chamber. The aminosilanized coverslips were stored at room temperature in a closed container for up to 2 weeks.
Vectabond
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom
Vectabond is a laboratory reagent used to facilitate adhesion of tissue sections to microscope slides. It is a silane-based formulation that provides a covalent bond between the glass surface and the tissue section, ensuring secure attachment during immunohistochemical and other staining procedures.
Automatically generated - may contain errors
Lab products found in correlation
Vectabond treatment, by Vector Laboratories (2 mentions)
Silicone templates, by Grace Bio-Labs (2 mentions)
Nhs ester peg, by Thermo Fisher Scientific (2 mentions)
Custom hybriwell chambers, by Grace Bio-Labs (2 mentions)
Dual silicon press fit tubing connectors, by Grace Bio-Labs (2 mentions)
Sodium bicarbonate, by Merck Group (2 mentions)
5kda mpeg succinimidyl carbonate, by Laysan Bio (2 mentions)
Neurobiotin, by Vector Laboratories (2 mentions)
Tetramethyl rhodamine labeled dextran, by Thermo Fisher Scientific (2 mentions)
Streptavidin cy2 conjugate, by Jackson ImmunoResearch (2 mentions)
C1si fast spectral confocal system, by Nikon (2 mentions)
Alexa fluor 555 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Avidin biotin blocking kit, by Vector Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Mouse irrelevant igg1, by BD (2 mentions)
Biotin anti human ctla4 cd152 clone bni3, by BD (2 mentions)
Pre chilled methyl butane, by Merck Group (2 mentions)
Circular coverslips, by Thermo Fisher Scientific (1 mentions)
Anti gfp a 11122, by Thermo Fisher Scientific (1 mentions)
Cy2 goat anti rabbit, by Jackson ImmunoResearch (1 mentions)
40 protocols using vectabond
1
Aminosilanization of Glass Coverslips
2
Coverslip Functionalization for Single-Molecule Imaging
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Borosilicate glass coverslips (1.5 MenzelGläzer, Germany) were heated to 500 °C in oven for 1 h to reduce background fluorescence. The coverslips were then rinsed with HPLC-grade acetone and immerged into 1% (v/v) Vectabond (product code #SP-1800, Vector Labs, CA, USA) in acetone for 10 min to functionalise the glass surface with amino groups. Coverslips were then rinsed with acetone followed by deionized water before drying them under a stream of nitrogen gas. A silicone gasket (103280, Grace Bio-Labs, OR, USA) with four reaction wells was placed in the middle of the coverslip. The coverslip surface was then simultaneously passivated by pegylation against unspecific protein/DNA binding and biotinylated to provide attachment points for specific protein immobilisation. Each well on the coverslip was thus filled with 20 µl of 180 mg/ml methoxy-PEG (5 kDa)-SVA (Laysan Bio, AL, USA) and 4.4 mg/ml biotin-PEG (5 kDa)-SC (Laysan Bio, AL, USA) in 50 mM MOPS-KOH buffer (pH 7.5), incubated for ∼ 3 h at room temperature and finally the wells were thoroughly rinsed with phosphate-buffered saline (PBS; Sigma Aldrich, UK). The coverslips remained functional for at least two weeks when stored at 4 °C in plastic pipette tip box containing a layer of deionised water at the bottom. During the storage the coverslip wells were filled with PBS.
3
PEGylation of Glass Coverslips for Flow Chambers
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Plasma cleaned glass coverslips (22 × 22 mm No. 1) were aminosilanized through Vectabond treatment (Vectabond in acetone 2% vol/vol; Vector Laboratories). Silicone templates (Grace bio-Labs) were used to treat a small section of the coverslips with a PEG solution containing 5kDa biotin-terminated PEG (2.5% w/w in molecular biology grade (MB) water, NOF Corp.), and 5kDa mPEG succinimidyl carbonate (25% w/w in MB water, Laysan Bio Inc.) in 0.1M sodium bicarbonate (Sigma-Aldrich). The coverslips were then left to incubate in a dark and moist environment overnight. On the day of the experiment, the coverslips were treated with another round of PEGylation with a short chain 333 Da NHS-ester PEG (Thermo Scientific) and incubated for 2–3 hrs. After washing off excess PEG, the coverslips were dried with a mild flow of nitrogen. Custom hybriwell chambers (Grace bio-Labs) with dual silicon press-fit tubing connectors (Grace bio-Labs) were placed atop the coverslips to construct a flow chamber.
4
Dye Injection and Immunohistochemistry for Neuronal Labeling
5
Cryosectioning of Biofilm Samples
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The biofilms grown on polycarbonate membranes by colony-biofilm method were carefully covered with a layer of optimum cutting temperature (OCT formulation, Tissue-Tek®, Sakura, Alphen aan den Rijn, The Netherlands) and placed in dry ice until completely frozen. The frozen samples were dipped vertically into the center of a cryosectioning mold filled with fresh OCT. The frozen samples were sectioned at −19 °C using a CM1850 cryostat (Leica, Wetzlar, Germany). The 5-μm thick cryosections were mounted on glass slides treated by Vectabond (Vector laboratories, Peterborough, UK).
6
Immunocytochemical Analysis of Retinal Cell Receptors
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Retinal cells were isolated as previously described [33 (link)]. Briefly, mouse retinas were prepared in sterile 0.9% saline. Approximately 200 mg of retinal tissue was incubated with saline containing 0.4 mg/ml papain for 30 min at 37 °C. The tissues were triturated with a siliconized pipette and dissociated single cells were centrifuged at 500 × g for 10 min. The cells were fixed in 4% paraformaldehyde for 10 min, centrifuged, and washed with saline. The cells were then placed onto a Superfrost Plus slide (Fisher Scientific) treated with Vectabond (Vector Labs) using a Cytospin4 (Thermoscientific) and stored at −80 °C until used for immunocytochemistry. For immunocytochemistry, the cells were permeabilized in methanol, incubated with 1% SDS in 0.01M PBS, and washed in 1X PBS. The slides were then incubated with 1% sodium borohydride, blocked using the mouse on mouse kit (Vector Labs), and colabeled with GS and the BMP receptor antibodies (BMPR1A, BMPRIB, and activin-like kinase receptor 2 [ALK2]). Following overnight incubation with the primary antibodies, the slides were incubated with the appropriate secondary antibodies with biotin streptavidin amplification used for the receptor antibodies. The slides were then washed, counterstained with Hoechst solution, and mounted with Aqua polymount.
7
Vectabond Pretreatment for CODEX Assays
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For CODEX assays, square (22 x 22 mm) glass coverslips (72204-10, Electron Microscopy Sciences) were pre-treated with Vectabond (Vector Labs) according to the manufacturer’s instructions. Briefly, using glass beakers, coverslips were immersed in 100% acetone for 5 min and then incubated in a mixture of 7 mL Vectabond and 350 mL 100% acetone for 30 min. Coverslips were washed in 100% acetone for 30 seconds, air-dried, baked at 70°C for 1 hour, and stored at room temperature. FFPE blocks were sectioned on Vectabond-treated coverslips and stored in a coverslip storage box (CS-22, Qintay, LLC) at 4°C in a vacuum desiccator (Thermo Fisher) containing drierite desiccant (07-578-3A, Thermo Fisher) until use for CODEX experiments.
8
Skeletal Muscle Fiber Type Analysis
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Cross-sections (10 μm thick) were cut from extensor digitorum longus (EDL) and soleus rat muscles. The cross-sections were mounted on Vectabond (Vector Laboratories, Burlingame, CA, USA) coated slides and stored in −80 °C until further analysis. To determine the different fiber types in each rat skeletal muscle, images were captured from adenosinetriphosphatase (ATPase) stained cross-sections following the methods described in [64 (link),65 (link)]. Digital pictures of cross-sections were captured using a 20× objective (DMRB microscope, Leica, Wetzlar, Germany) to finally differentiate between the four fiber types (type I, type IIA, type IIX, and type IIB [7 (link)].
9
Characterizing Graft-Infiltrating Immune Cells
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Tissues were collected from one recipient with no DCreg infusion (control group) on the day of euthanasia and from one recipient with DCreg infusion (experimental group) on POD 28 by open biopsy of the kidney graft. Tissues were embedded in O.C.T. (Miles), snap-frozen, and stored at −80°C. Cryostat sections (8–10 µm) were mounted on slides pre-coated with Vectabond (Vector) then fixed in 96% ethanol and allowed to dry. Sections were blocked successively with 5% goat serum and an avidin/biotin blocking kit (Vector). Next, sections were incubated with anti-human CD4 Ab (Dako; Clone 4B12, 1:100, overnight, 10°C), followed by Alexa Fluor 555-goat anti-mouse IgG (Molecular Probes, 1:400, 1 h, RT). The slides were then blocked with mouse irrelevant IgG1 (BD Biosystems, 1:100, 1 h, RT) and incubated successively with biotin anti-human CTLA4 (CD152) (clone BNI3, BD Biosystems, 1:100, 1 h, RT), followed by Streptavidin Dylight 488 (Jackson Immunoresearch, 1:400, 1 h, RT). Cell nuclei were stained with DAPI (Molecular Probes).
10
Histological Analysis of Mouse Ear Sections
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