Tissue slides were deparaffinized, rehydrated, and placed in antigen unmasking solution (Vector; Burlingame, CA), then washed and permeabilized with 0.5% Triton. Slides were blocked in 4% Bovine Serum Albumin (BSA, Equitech-Bio, Kerrville, TX) with 0.1% Tween 20 in PBS for 1 h. Primary antibodies were diluted 1:50 with 1% BSA with 0.1% Tween 20 in PBS, placed on the slides and incubated overnight at 4 °C. The following primary antibodies were used: GFP Tag polyclonal, Alexa Fluor 488, secondary fluorescent antibodies, applied for 1 h were, Alexa Fluor 488 donkey or Alexa Fluor 594 goat anti-rabbit (Invitrogen, Eugene, OR), then washed in PBS and coverslipped using Vectashield Hard Set with 4′,6-diamidino-2-phenylindole (DAPI, Vector, Burlingame, CA). A negative control sample was incubated with no primary antibody. All slides were imaged on an Eclipse E800 microscope (Nikon, Melville, NY) with an Evolution QEi Monochrome camera with LCD color filter (Q-imaging, Canada).
Vectashield hardset with 4 6 diamidino 2 phenylindole dapi
Manufactured by Vector Laboratories
Sourced in Canada
Vectashield Hardset with DAPI is a mounting medium designed for fluorescence microscopy. It contains the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI), which selectively binds to DNA and emits blue fluorescence when excited. This product is intended to be used for the preservation and visualization of fluorescently labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Equitech-Bio (2 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Antigen unmasking solution, by Vector Laboratories (1 mentions)
Eclipse e800 microscope, by Nikon (1 mentions)
Plan achromat, by Olympus (1 mentions)
Ckx41, by Olympus (1 mentions)
Ep888y, by Abcam (1 mentions)
Goat anti rabbit af488, by Thermo Fisher Scientific (1 mentions)
Apo brdu tunel assay kit, by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Fv10i confocal microscope, by Olympus (1 mentions)
Alexa 488 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bassoon monoclonal antibodies, by Enzo Life Sciences (1 mentions)
5 protocols using vectashield hardset with 4 6 diamidino 2 phenylindole dapi
1
Immunofluorescence Staining of GFP Samples
2
Immunohistochemical Analysis of Liver Tissue
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Liver tissue from BALB/c or NSG mice was embedded in OCT (CellPath) and frozen in isopentane on dry ice. 8 µm sections were cut and transferred to Superfrost plus slides (Thermo Scientific), air dried overnight and fixed in 100% acetone. Following blocking, primary antibodies against FcRn or FcγRII were added overnight before detection with Alexafluor 488-labeled secondary antibody (online supplementary table 1 ) for 45 min. Subsequently, primary antibodies against Clec4F or cytokeratin 8 were added for 2 hours before detection with AlexaFluor 549 or AlexaFluor 568 conjugated secondary antibodies (online supplementary table 1 ). Slides were mounted using Vectashield hardset with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories).
Images were collected using a CKX41 inverted microscope with a reflected fluorescence system equipped with a DP22 camera running CellSens software, using Plan Achromat 10×0.25 and 40×0.65 objective lenses (all from Olympus). Images were transferred to ImageJ (Fiji) or photoshop (Adobe) where background autofluorescence was removed, contrast stretched and brightness adjusted to maximize clarity, with all images treated equivalently.
Images were collected using a CKX41 inverted microscope with a reflected fluorescence system equipped with a DP22 camera running CellSens software, using Plan Achromat 10×0.25 and 40×0.65 objective lenses (all from Olympus). Images were transferred to ImageJ (Fiji) or photoshop (Adobe) where background autofluorescence was removed, contrast stretched and brightness adjusted to maximize clarity, with all images treated equivalently.
3
Immunofluorescent Staining of Frozen Tissue
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Tissues were frozen in OCT media (Cell Path) and placed in isopentane on a bed of dry ice. 10 μm frozen sections were then cut, fixed in acetone, and blocked with 5% normal goat serum before incubation with mAb to hFcγRIIB (EP888Y, Abcam), mFcγRII (AT130-5, in-house) or B cells (B220, BD Pharmingen) followed by goat anti-human-AF488 (Invitrogen), goat anti-rabbit-AF488 (Invitrogen) or goat anti-rat-AF647 (Invitrogen). Slides were mounted using Vectashield hardset with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Images were collected using a CKX41 inverted microscope using a Plan Achromat 10×0.25 objective lens (Olympus). RGB images (TIFF) were transferred to Adobe Photoshop CS6 and RGB image overlays created. Background autofluorescence was removed, contrast stretched, and brightness adjusted to maximize clarity, with all images treated equivalently.
4
Apoptosis Assessment in Cell Cultures
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Cells (100,000/well) plated in eight-chambered glass slides coated with poly-d -lysine (Sigma-Aldrich) were fixed in 4% paraformaldehyde (10 min, room temperature). Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method using APO-BrdUTM TUNEL Assay Kit (InvitrogenTM, Life Technologies). Staining for cleaved caspase-3 with primary antibody (rabbit, 1:100, Cell Signaling Technology, Beverly, MA, USA; 4 °C, overnight) was followed by Alexa488-conjugated secondary antibody (1:500, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Slides were mounted with Hardset Vectashield (with DAPI (4′,6-diamidino-2-phenylindole)) (Vector Laboratories, Burlington, ON, Canada) and imaged using Olympus FV10i confocal microscope.
5
Immunolabeling Neuronal Markers
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Bassoon monoclonal antibodies were obtained from Enzo Lifescience, GFAP monoclonal antibodies were obtained from Invitrogen, calbindin polyclonal antibody from Invitrogen, synaptophysin antibody from Sigma and secondary antibodies conjugated with AlexaFluor 488, 550 and 633 were obtained from Thermo sher. Hardset Vectashield™ with DAPI (4′,6-diamidino-2-phenylindole) was obtained from Vector Laboratories.
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