Brains, midguts, and abdomens containing fat bodies were dissected in PBS and fixed for 15 min in PBS containing 4% paraformaldehyde. After fixation, the samples were washed with PBS containing 0.2% Triton X-100 (PBST) and blocked with 1% BSA in PBST for 30 min. After incubation with primary antibodies overnight at 4°C: p-ERK (1:100, Cell Signaling, 4370), Prospero (1:100, DSHB, MR1A), ILP2 (1:1000, a kind gift from Hugo Stocker), or Pvf1 (1:50, a kind gift from Ben-Zion Shilo). Tissues were washed and then incubated with secondary antibody and DAPI for 1h, washed, and mounted in Vectashield (Vector). C2C12 myoblasts were cultured and differentiated on cover slides. Treated C2C12 myotubes were washed and fixed for 15 min in PBS containing 4% formaldehyde. After fixation, the samples were washed with PBST, blocked with 1% BSA in PBST, and incubated with primary antibody against MHC (1:50, DSHB, MF20) overnight at 4°C. Cells were then incub ated with secondary antibody and DAPI for 1h, washed and mounted in Vectashield (Vector). Treated 3T3-L1 mature adipocytes were incubated with Bodipy 493/503 (1 μg/mL, Life Technologies, D3922) for 20 min, washed, and imaged. Regular microscopy was performed on a Zeiss Axioskop 2motplus or a Nikon SMZ18 and confocal images were obtained using a Leica system.
Vectashield
Manufactured by Vector Laboratories
Sourced in United States, United Kingdom, Germany, Canada, Japan, France, Switzerland, Panama, Spain, China, Italy
Vectashield is a non-hardening, aqueous-based mounting medium designed for use with fluorescent-labeled specimens. It is formulated to retard photobleaching of fluorescent dyes and provides excellent preservation of fluorescent signals.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (157 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (146 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (140 mentions)
Triton x 100, by Merck Group (87 mentions)
Lsm 700, by Zeiss (81 mentions)
Lsm 700 confocal microscope, by Zeiss (78 mentions)
Lsm 780, by Zeiss (73 mentions)
Lsm 710 confocal microscope, by Zeiss (73 mentions)
Bovine serum albumin (bsa), by Merck Group (71 mentions)
Hoechst 33342, by Thermo Fisher Scientific (70 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (57 mentions)
Lsm 510, by Zeiss (55 mentions)
Alexa 488, by Thermo Fisher Scientific (47 mentions)
Lsm 710, by Zeiss (45 mentions)
Lsm 880, by Zeiss (44 mentions)
Paraformaldehyde (pfa), by Merck Group (42 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (39 mentions)
To pro 3, by Thermo Fisher Scientific (39 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (38 mentions)
Lsm 780 confocal microscope, by Zeiss (35 mentions)
3 780 protocols using vectashield
1
Immunostaining for Signaling and Morphology
2
Nuclei Spread Preparation for Microscopic Analysis
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Rosette leaf material was harvested and directly fixed in ice-cold Carnoy’s fixative (3:1 ethanol/acetic acid) and kept at –20° until use. Nuclei spread preparations for microscopic analysis were made essentially as described by Schubert et al. (2001 (link)) and Tessadori et al. (2009 (link)) using modified enzymatic cell wall–degrading mixture [cellulose Onozuka R10 (Yakult), 0.25% macerozyme R10 (Duchefa) in 10 mM citrate buffer, pH 4.5] to digest cell walls. The air-dried slides were mounted in Vectashield (Vector Laboratories) with DAPI (2 µg ml-1) before observation and capturing.
For the (de)etiolation experiments, cotyledons of 5-d-old seedlings were fixed in 4% paraformaldehyde for 3 hr under white light condition or under a safe green light for the dark-grown seedlings, and treated with a solution containing 0.5% cellulose Onozuka R10 (Yakult), 0.25% macerozyme R10 (Duchefa), and 0.1% Triton X-100 for 1 hr. Cotyledons from at least three seedlings were isolated and squashed on a glass slide, flash frozen in liquid nitrogen, and incubated with PEMSB (50 mM Pipes, pH 7.3; 5 mM EGTA, pH 7.1; 5 mM MgSO4; 0.05% saponin; 5% wt/vol BSA) before being mounted with Vectashield (Vector Laboratories) supplemented with 2 μg/ml DAPI before observation and capturing.
For the (de)etiolation experiments, cotyledons of 5-d-old seedlings were fixed in 4% paraformaldehyde for 3 hr under white light condition or under a safe green light for the dark-grown seedlings, and treated with a solution containing 0.5% cellulose Onozuka R10 (Yakult), 0.25% macerozyme R10 (Duchefa), and 0.1% Triton X-100 for 1 hr. Cotyledons from at least three seedlings were isolated and squashed on a glass slide, flash frozen in liquid nitrogen, and incubated with PEMSB (50 mM Pipes, pH 7.3; 5 mM EGTA, pH 7.1; 5 mM MgSO4; 0.05% saponin; 5% wt/vol BSA) before being mounted with Vectashield (Vector Laboratories) supplemented with 2 μg/ml DAPI before observation and capturing.
3
Immunostaining of Activated Leukocyte Adhesion
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Antibody staining was performed as previously described [46 (link),47 (link)]. Primary antibodies were used at the following dilutions: mouse anti- activated leukocyte cell adhesion molecule (ALCAM/zn5; ZIRC) 1:50, rabbit anti-DsRed (AnaSpec, Fremont, CA) at 1:200 in phosphate buffered saline with 0.03% triton and 4% bovine serum albumin (PBT). Secondary anti mouse antibodies (Alexa 488, Alexa 568; Invitrogen) were used at 1:200 dilution in PBT. Embryos were mounted in Vectashield or Vectashield with Dapi (Vector Laboratories). Confocal images were collected on an Olympus Fluoview FV1000 microscope. Brightest point projections were made using Olympus Fluoview software and images were processed using Adobe Photoshop. Optical sections in z-series were collected at 0.52 μm intervals.
4
Immunofluorescence Staining of Tissue Sections and Cultured Cells
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Immunofluorescence staining was performed in fresh frozen OCT tissue sections with primary antibodies against ATP7A (LS Bioscience)(1:300) and CD31 (BD Bioscience) (1:300). Secondary antibodies were Alexa Fluor 488 or 546-conjugated goat anti-mouse IgG and goat anti-rat IgG (Invitrogen)(1:500). Tissue sections were mounted using Vectashield (Vector Laboratories) containing DAPI for nuclear counter-staining. For cultured cells, HUVECs on glass coverslips were rinsed quickly in ice-cold PBS, fixed in freshly prepared 4% paraformaldehyde in PBS for 10 min at room temperature, permeabilized in 0.05% Triton X-100 in PBS for 5 min, and rinsed sequentially in PBS, 50 μmol/L NH4Cl and PBS for 10 min each. After incubation for 1 h in blocking buffer (PBS + 3%BSA), cells were incubated with primary antibody for 18 h at 4 °C, rinsed in PBS/BSA, and then incubated in Alexa Fluor 488-conjugated IgG for 1 h at room temperature, and cells rinsed with PBS. Cells on coverslips were mounted onto glass slides using Vectashield (Vector Laboratories) and observed using confocal microscopy.
5
Tissue Preparation for Retinal Analysis
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Retrieved eyecups (>6 weeks after injections) were fixed in 4% paraformaldehyde (PFA) in PBS for 24 hr at 4°C after the cornea and lens had been removed anteriorly under a light microscope. The tissue was then washed in PBS prior to incubation in PBS containing 30% sucrose overnight at 4°C. For whole mounts, fixed eyes were washed in PBS, and whole retinas were carefully dissected under a light microscope. Retinas were then flat-mounted with fluorescent mounting medium containing DAPI (Vectashield, Vector Laboratories, Peterborough, UK) to stain cell nuclei. For cryosections, fixed eyes were cryo-protected in optimal cutting temperature medium (Raymond A. Lamb, Eastbourne, UK) and frozen at −80°C until further processing. The cryo-protected retina was sectioned (generally 8–10 μm thickness) on a cryostat (Leica Microsystems) horizontally through the eyecup from ventral to dorsal sides so that each section contained a complete nasal to temporal cross-section of the retina. Slides were stored at −80°C. Prior to analysis, the slides were removed from the freezer, allowed to air-dry at room temperature for 1 hr, and mounted with fluorescent mounting medium containing DAPI (Vectashield, Vector Laboratories, Peterborough, UK) to stain cell nuclei.
6
Immunostaining and F-actin Visualization
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For immunostaining, we followed standard procedures (Williams et al., 2014 (link)). For F-actin staining, Alexa-coupled Phalloidin (1:200; Invitrogen) was added in PBS/2% Tween-20 for 30 min prior to final washes and mounting. The samples were mounted in Vectashield (Vector)+DAPI, or incubated with the DNA dye TOPRO-3 (1:1000; Molecular Probes) for 10 min and then mounted in Vectashield (Vector).
7
Dissection and Fixation of Drosophila Nervous System
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Dissection was performed in phosphate-buffered saline (PBS), and larvae were fixed in 4% paraformaldehyde (PanReac AppliChem, Spain) for 20 min. Then, samples were washed three times in PBS for 5 min and stored in an antifade mounting medium for fluorescent samples (Vectashield, Vector Laboratories, USA).
Brain and ventral nerve cords of adult flies were dissected and fixed in freshly prepared 4% paraformaldehyde for 10 min. Next, they were washed in PBS 3 × 5 min and placed in a medium for fluorescent microscopy (Vectashield, Vector Laboratories, USA).
Brain and ventral nerve cords of adult flies were dissected and fixed in freshly prepared 4% paraformaldehyde for 10 min. Next, they were washed in PBS 3 × 5 min and placed in a medium for fluorescent microscopy (Vectashield, Vector Laboratories, USA).
8
Immunofluorescence Staining of Respiratory Structures
9
Intracranial Nanotracer Injections in Mice
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Experiments were approved by the Northwestern University Animal
Care
and Use Committee. Injections were performed in adult C57BL/6J mice
under inhaled anesthesia (1–2% isoflurane in 0.5 L/min O2). After a midline skin incision and alignment of the skull,
a small (0.5–1.0 mm) craniotomy was performed and a glass micropipette
containing the nanotracers lowered into the brain to reach the desired
stereotaxic location of the injection site (CA1:1.8 mm lateral, 2.3
mm caudal of bregma, 1.4 mm deep from dura; V1:2.5 mm lateral, 3.3
mm caudal of bregma, and 0.5 mm deep from dura). A calibrated volume
(typically 30 nL) of solution was injected by applying positive pressure
(typically 1–2 psi). After withdrawing the micropipette, the
craniotomy was filled with Kwik-Sil (WPI) and the incision site closed
with 5–0 silk suture. After 48–72 h, mice were deeply
anesthetized and perfused with 4% paraformaldehyde (PFA). Brains were
set in PFA overnight and then transferred to 30% sucrose in PBS for
∼3 days before 50 μm thick sagittal (for CA1 injections)
or coronal (for V1 injections) sections were cut with a microtome.
Slices were mounted on glass slides using Vectashield (Vector Laboratories)
with DAPI, except for Coumarin injections, in which case slices were
instead placed in propidium iodide in PBS for 3–5 min before
mounting with Vectashield, without DAPI.
Care
and Use Committee. Injections were performed in adult C57BL/6J mice
under inhaled anesthesia (1–2% isoflurane in 0.5 L/min O2). After a midline skin incision and alignment of the skull,
a small (0.5–1.0 mm) craniotomy was performed and a glass micropipette
containing the nanotracers lowered into the brain to reach the desired
stereotaxic location of the injection site (CA1:1.8 mm lateral, 2.3
mm caudal of bregma, 1.4 mm deep from dura; V1:2.5 mm lateral, 3.3
mm caudal of bregma, and 0.5 mm deep from dura). A calibrated volume
(typically 30 nL) of solution was injected by applying positive pressure
(typically 1–2 psi). After withdrawing the micropipette, the
craniotomy was filled with Kwik-Sil (WPI) and the incision site closed
with 5–0 silk suture. After 48–72 h, mice were deeply
anesthetized and perfused with 4% paraformaldehyde (PFA). Brains were
set in PFA overnight and then transferred to 30% sucrose in PBS for
∼3 days before 50 μm thick sagittal (for CA1 injections)
or coronal (for V1 injections) sections were cut with a microtome.
Slices were mounted on glass slides using Vectashield (Vector Laboratories)
with DAPI, except for Coumarin injections, in which case slices were
instead placed in propidium iodide in PBS for 3–5 min before
mounting with Vectashield, without DAPI.
10
Chromosome Counting in Potato Root Tips
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Root tips of 1–2 cm in length were harvested from young plants grown in vitro on MS medium (Murashige and Skoog, 1962 (link)) containing 1 mg L–1 NAA, fixed in ethanol/acetic acid (3:1) and stored in 70% ethanol at 4°C. The root tips were washed twice in distilled water (10 min each), digested with a 1% Cellulase Onozuka R-10, 0.4% Cytohelicase and 0.4% Pectolyase in citrate buffer at pH = 4.8 (30 min) and squashed in 45% acetic acid. After the cover slips were removed in liquid nitrogen, slides were dehydrated by placing them sequentially in 70, 90, and finally 100% ethanol. Chromosomes were stained using 4′,6–diamidine-2′-phenylindolehydrochloride (DAPI) and washed with 2xSSC and distilled water. Probes were mounted in Vectashield (Vectashield, Vector Laboratories) to preserve the staining. For chromosome counts, at least five cells with well-spread metaphase chromosomes were used for each clone. Best spreads were photographed with a digital camera attached to an epifluorescent microscope (Olympus BX 60 with appropriate filter for DAPI). For all cytogenetic evaluations of the parental lines, the diploid blb41 and tetraploid cultivated potatoes were used as internal standards.
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