For actin staining, NIH 3T3 cells (5 × 104) were seeded in 35 mm glass bottom dishes coated with 0.002% PLL. After 18 h of cultivation, cells were washed with PBS and 1 ml serum free DMEM containing liposomes (final lipid concentration 25 μM) or anti-luciferase siRNA (100 pmol) was added before the LET with a constant current of 0.34 mA cm−2 for 15 min. Immediately after LET, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, cells were permeabilized with 1% Triton X-100 at room temperature for 10 min. The cells were then incubated with rhodamine phalloidin at a final concentration of 100 nM for 30 min in dark. After washing, cells were mounted in VECTASHIELD with DAPI and observed by CLSM A1R+ (Nikon Co., Ltd., Japan).
Vectashield
Manufactured by Nikon
Sourced in Japan
Vectashield is a mounting medium developed for fluorescence microscopy. It is designed to preserve the fluorescence signal of samples while providing a suitable environment for their visualization and imaging.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Lsm 5 pascal, by Zeiss (1 mentions)
Ec plan neofluar 20, by Zeiss (1 mentions)
Bz 8000, by Keyence (1 mentions)
Elements ar, by Nikon (1 mentions)
Ti microscope, by Nikon (1 mentions)
Anti mouse alexafluor 647, by Jackson ImmunoResearch (1 mentions)
Axio imager z1 fluorescence microscope, by Zeiss (1 mentions)
Mouse anti flag m2, by Merck Group (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Ti2 e microscope, by Nikon (1 mentions)
5 protocols using vectashield
1
Actin Staining of NIH 3T3 Cells
2
Comprehensive Microscopic Analysis of Tissue Perfusion
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To assess vessel perfusion, the hypoxic areas, apoptosis, and pericyte coverage, cryosections were processed and were visualized and imaged using an all-in-one microscope system (BZ8000; Keyence Corporation, Osaka, Japan) with a 20× objective lens (Plan Fluor 20×: numerical aperture [NA] = 0.75; working distance = 1.0 mm; Nikon Corporation, Tokyo, Japan).
The infarct area that was stained with rhodamine-conjugated G. simplicifolia lectin I was placed on a microscope slide with Vectashield® and it was flattened with a cover slip. New vessels in the infarct area were imaged using confocal microscopy (LSM 5 Pascal; Carl Zeiss AG, Oberkochen, Germany) by scanning 12 layers (with 2.5 μm between the adjacent layers) using a 20 × objective lens (EC Plan-Neofluar 20 × : NA = 0.5; working distance = 2.0 mm; Carl Zeiss AG).
The infarct area that was stained with rhodamine-conjugated G. simplicifolia lectin I was placed on a microscope slide with Vectashield® and it was flattened with a cover slip. New vessels in the infarct area were imaged using confocal microscopy (LSM 5 Pascal; Carl Zeiss AG, Oberkochen, Germany) by scanning 12 layers (with 2.5 μm between the adjacent layers) using a 20 × objective lens (EC Plan-Neofluar 20 × : NA = 0.5; working distance = 2.0 mm; Carl Zeiss AG).
3
Kinetoplast Staining and Microscopy Protocol
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For experiments with kinetoplast staining, MitoTracker® Red was added to the culture at 500 nM and shaken at 27 °C for 30 minutes to allow uptake. Cells were harvested for microscopy at 2–5 × 106cells/mL and washed twice in PBS before suspending in 3.7% formaldehyde/PBS solution and 100 μl of suspension was fixed to glass slides for 10 minutes. Cells were washed three times in PBS, permeabilized with 0.1% Triton X-100 and washed an additional three times in PBS. Blocking was performed for 60 minutes using 5.5% FBS supplemented with 0.05% Tween 20 in PBS. All incubation steps were performed in humid chamber and all antibodies were used at concentrations indicated in text. Cells were stained using 100 uL 1 μg/mL DAPI solution for 1 minute and washed three times with PBS. Cells were air dried and mounted using Vectashield® before imaging was performed using a Nikon Ti microscope. Image analysis was performed using ImageJ (NIH) and Nikon Elements AR.
4
Immunofluorescence Imaging of Transfected AGS Cells
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Transfected AGS cells were fixed with 4% paraformaldehyde in PBS 1x (Gibco, Carlsbad, CA, USA) for 20 min at room temperature and washed three times with PBS 1x. The cells on coverslips were mounted on glass slides using Vectashield (Vector Laboratories, Newark, CA, USA). AGS cells transfected with GFP-plasmids were directly observed with the microscope, while AGS cells transfected with the CagA1-1216 construct, after fixation, were blocked with 1% BSA in PBS 1x for 30 min, then incubated with mouse anti-CagA (Sc-28368; Santa Cruz, CA, USA) for 1 h at 37°C, followed by incubation with anti-mouse Alexa Fluor 647 (#715-605-150; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 40 min at room temperature. Cells on coverslips were mounted on glass slides using Vectashield.. Images were acquired with a Nikon Eclipse Ti confocal laser scanning microscope (Nikon Corp) with a 20x objective.
5
Immunofluorescence of Parasite-Infected Fibroblasts
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Human foreskin fibroblasts (HFF) were grown on coverslips in 24-well plates until confluent and were infected with parasites. The cells were rinsed once with phosphate-buffered saline (PBS), fixed with 3.7% formaldehyde in PBS, washed, permeabilized with 0.1% Triton X-100, blocked with 3% bovine serum albumin (BSA) for 1 h, and incubated with primary antibodies for a minimum of 2 h. Secondary antibodies used were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Thermo Scientific). The coverslips were mounted in Vectashield (Vector Labs) and viewed with an Axio Imager.Z1 fluorescence microscope (Zeiss). For proximity-ligation assays, cells were fixed in 4% paraformaldehyde/4% sucrose followed by permeabilization in 0.1% Triton X-100 for 10 min. Blocking, antibody incubations, and proximity ligation were conducted according to the manufacturer’s directions (Sigma-Aldrich) using rabbit polyclonal anti-HA (63 (link)) and mouse m2 anti-FLAG (Sigma-Aldrich; F1804) as primary antibodies. Cells were counterstained with Alexa Fluor 647-conjugated rabbit anti–β-tubulin, mounted in Vectashield, and imaged on a Nikon Ti2E microscope.
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